Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1ß (IL-1ß). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1ß. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

Prostaglandin pathway gene expression in human placenta, amnion and choriodecidua is differentially affected by preterm and term labour and by uterine inflammation.

BACKGROUND: Elucidation of the biochemical pathways involved in activation of preterm and term human labour would facilitate the development of effective management and inform judgements regarding the necessity for preterm tocolysis and post-term induction. Prostaglandins act at all stages of human reproduction, and are potentially activators of labour. METHODS: Expression of 15 genes involved in prostaglandin synthesis, transport and degradation was measured by qPCR using tissue samples from human placenta, amnion and choriodecidua at preterm and full-term vaginal and caesarean delivery. Cellular localisation of eight prostaglandin pathway proteins was determined by immunohistochemistry. RESULTS: Expression of prostaglandin pathway genes was differentially affected by factors including gestational age at delivery, and the incidence and duration of labour. Chorioamnionitis/deciduitis was associated with upregulation of PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), along with the inflammatory genes IL8 (interleukin 8), S100A8 (S100 calcium binding protein A8) and TLR2 (toll-like receptor 2), in amnion and choriodecidua, and with downregulation of CBR1 (carbonyl reductase 1) and HPGD (hydroxyprostaglandin dehydrogenase 15-(NAD)) in choriodecidua. Protein localisation differed greatly between the various maternal and fetal cell types. CONCLUSIONS: Preterm and term labour are associated with distinct prostaglandin pathway expression profiles; inflammation provokes specific changes, unrelated to the presence of labour; spontaneous and induced term labour are indistinguishable.

Expression of genes related to prostaglandin synthesis or signaling in human subcutaneous and omental adipose tissue: depot differences and modulation by adipogenesis.

OBJECTIVES: (1) To examine depot-specific PGE2 and PGF2α release and mRNA expression of enzymes or receptors involved in PG synthesis or signaling in human adipose tissues; (2) to identify changes in expression of these transcripts through preadipocyte differentiation; and (3) to examine associations between adipose tissue mRNA expression of these transcripts and adiposity measurements. METHODS: Fat samples were obtained surgically in women. PGE2 and PGF2α release by preadipocytes and adipose tissue explants was measured. Expression levels of mRNA coding for enzymes or receptors involved in PG synthesis or signaling were measured by RT-PCR. RESULTS: Cultured preadipocytes and explants from omental fat released more PGE2 and PGF2α than those from the subcutaneous depot and the corresponding transcripts showed consistent depot differences. Following preadipocyte differentiation, expression of PLA2G16 and PTGER3 mRNA was significantly increased whereas COX-1, COX-2, PTGIS, and PTGES mRNA abundance were decreased in both compartments (P ≤ 0.01 for all). Transcripts that were stimulated during adipogenesis were those that correlated best with adiposity measurements. CONCLUSION: Cells from the omental fat compartment release more PGE2 and PGF2α than those from the subcutaneous depot. Obesity modulates expression of PG-synthesizing enzymes and PG receptors which likely occurs through adipogenesis-induced changes in expression of these transcripts.

Evaluation of the prostaglandin F synthase activity of human and bovine aldo-keto reductases: AKR1A1s complement AKR1B1s as potent PGF synthases.

AKR1B1 of the polyol pathway was identified as a prostaglandin F2α synthase (PGFS). Using a genomic approach we have identified in the endometrium five bovine and three human AKRs with putative PGFS activity and generated the corresponding recombinant enzymes. The PGFS activity of the recombinant proteins was evaluated using a novel assay based on in situ generation of the precursor of PG biosynthesis PGH2. PGF2α was measured by ELISA and the relative potencies of the different enzymes were compared. We identified AKR1A1 and confirmed AKR1B1 as the most potent PGFS expressing characteristic inhibition patterns in presence of methylglyoxal, ponalrestat and glucose.

Differential expression of the enzymatic system controlling synthesis, metabolism, and transport of PGF2 alpha in human fetal membranes.

The present study investigated the expression of genes and proteins associated with PGF2alpha biosynthesis, catabolism, and transport in matched amnion and choriodecidua of human term placenta. The concentration of PGF2alpha within fetal membranes depends on the balance between complex enzymatic systems responsible for, respectively, its synthesis-by prostaglandin-endoperoxide synthase 2 (PTGS2) and members of the aldo-keto reductase (AKR) family, AKR1C3 and AKR1B1-and its catabolic inactivation-through hydroxy-prostaglandin-dehydrogenase (HPGD). We observed that AKR1C3 shows equal basal expression (mRNA and protein) in choriodecidua and amnion but that AKR1B1 exhibits preferential expression in the choriodecidua. Expression of HPGD and solute carrier organic anion transporter family member 2A1 (SLCO2A1) was found primarily in the choriodecidua. We also evaluated whether an inflammatory environment induced by the gram-negative bacterial endotoxin lipopolysaccharide (LPS) affects expression of each candidate enzymes. The amnion responded to LPS with a small but significant decrease of AKR1B1 mRNA expression. In contrast, we found a significant increase in PTGS2 and AKR1C3 mRNA expression in choriodecidua after LPS challenge, but such regulation was confirmed only at protein levels for PTGS2 and not for AKR1C3. Our results suggest that the choriodecidua appears to be the main tissue, which expresses maximally all the components (synthesis, degradation, and transport) controlling PGF2alpha levels.

Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line.

In ruminants, interferon-tau (IFNtau) is the maternal recognition signal inhibiting prostaglandin (PG) F2alpha production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFNtau acts as a molecular switch that turns on PGE(2) production in endometrial stromal cells while suppressing PGF2alpha production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2alpha and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFNtau up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFNtau inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFNtau at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFNtau, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFNtau action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFNtau action and PG production in the bovine.

Role of Dicer1-Dependent Factors in the Paracrine Regulation of Epididymal Gene Expression.

Dicer1 is an endoribonuclease involved in the biogenesis of functional molecules such as microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs). These small non-coding RNAs are important regulators of post-transcriptional gene expression and participate in the control of male fertility. With the knowledge that 1) Dicer1-dependent factors are required for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1-dependent factors produced by the proximal epididymis (initial segment/caput)- including miRNAs- on the regulation of epididymal gene expression in the distal epididymis regions (i.e. corpus and cauda). To this end, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 35 and 33 transcripts that displayed significant expression level changes in the corpus and cauda regions (Fold change > 2 or < -2; p < 0.002), respectively. Among these transcripts, Zn-alpha 2-glycoprotein (Azgp1) encodes for a sperm equatorial protein whose expression in the epididymis of Dicer1 cKO mice is significantly increased compared to controls. In addition, 154 miRNAs, including miR-210, miR-672, miR-191 and miR-204, showed significantly impaired biogenesis in the absence of Dicer1 from the principal cells of the proximal epididymis (Fold change > 2 or < -2; p < 0.01). These miRNAs are secreted via extracellular vesicles (EVs) derived from the DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in distinct biological pathways, as evidenced by in silico analysis. Albeit correlative and based on in silico approach, our study proposes that Dicer1-dependent factors trigger- directly or not-significant genes expression changes in distinct regions of this organ. The paracrine control of functions important to post-testicular sperm maturation by Dicer1-dependent factors may open new avenues for the identification of molecular targets important to male fertility control.

Expression of human prostaglandin transporter in the human endometrium across the menstrual cycle.

Prostaglandins (PGs) are important regulators of reproductive function. The mechanism by which PGs are transported across the biological membrane is a new emerging field of investigation. Prostaglandin transporter (PGT) has been identified as a functional PG carrier. The aim of our study was to outline the expression of PGT in the human endometrium across the menstrual cycle. Quantitative RT-PCR showed human PGT (hPGT) expression to be strong in the proliferative and early secretory phases and low in the middle to late secretory phase. Northern blot analysis revealed hPGT mRNA transcript of 4 kb in the human endometrium. A peptide-directed polyclonal antibody was generated in rabbits against the 22 amino acids forming the C terminus of hPGT. Antibody specificity was demonstrated by Western blot. Immunoblots of endogenous hPGT in the human endometrium revealed a 70-kDa protein in endometrial cells. Endometrial biopsies collected across the menstrual cycle were used to assess hPGT protein expression by immunohistochemistry. hPGT was immunolocalized to luminal, glandular epithelial, and stromal cells. Because it was observed at the mRNA level, semiquantitative analysis showed a higher protein expression in proliferative and early secretory phases than in the mid-late secretory phase. In conclusion, our study revealed that hPGT expression is modulated in epithelial and stromal cells of the human endometrium at both mRNA and protein levels during the menstrual cycle. These findings support a role for hPGT as an important new player in the regulation of PG action in the human endometrium.

Molecular cloning and tissue distribution of microsomal-1 and cytosolic prostaglandin E synthases in macaque.

Prostaglandins derived from arachidonic acid are involved in a wide variety of physiological and pathological processes. The primary enzymes involved in the production of PGE2 from arachidonic acid are cyclooxygenases and prostaglandin E synthases. These enzymes have been identified in human, but only partially in the monkey where microsomal PGES-1 and cytosolic PGES have not been characterized. The present study was undertaken to clone these enzymes and to study their tissue distribution, along with mPGES-2. The coding sequence of Macaque mPGES-1 is 98% homologous to human mPGES-1 at the nucleic acid level and the deduced amino acid sequence has 98% homology with the human protein. The Macaque cPGES cDNA is more than 99% homologous to the human and the deduced amino acids sequence is identical to that of the human cPGES. By Northern blot analysis, we found that mPGES-2 and cPGES mRNA were expressed in the endometrium, myometrium, ovary and oviduct, albeit at different levels, while mPGES-1 mRNA was detected at a weak level, mainly in the oviduct. Western Blot analysis revealed that mPGES-2, mPGES-1 and cPGES proteins were present in all tissues tested. These results suggest that production of PGE2 in Macaque may involve more than one PGES and that further studies will be needed to fully understand the conditions under which each PGES contributes to PGE2 production.

Expression of microsomal prostaglandin E synthase in bovine endometrium: coexpression with cyclooxygenase type 2 and regulation by interferon-tau.

Prostaglandins (PGs) are important regulators of reproductive functions. In ruminants, interferon (IFN)-tau is the embryonic signal responsible for recognition of pregnancy. This is effected by a reduction of the production of PGF(2alpha) relative to PGE(2.) This may be accomplished by a decrease in PGF(2alpha) production, but a stimulation of PGE(2) via the PGE synthase might also be involved. The purpose of the present study was to confirm the presence of PGE synthase (PGES) in the bovine endometrium, identify the factors affecting its expression, and compare it with that of cyclooxygenase-2 (COX-2). This was done by Northern blot analysis using primary cultures of bovine epithelial and stromal cells of the endometrium and bovine endometrial cell line. PGES mRNA expression was increased in the presence of lipopolysaccharides, TNF-alpha, and IFN-tau in stromal cells and IFN-tau in epithelial cells. In stromal cells, IFN-tau induced a rapid increase of PGES and COX-2 mRNA expression. In bovine endometrial cells, phorbol 12-myristate 13-actetate increased PGES mRNA, COX-2 mRNA and PGE(2) production. These results suggest that in endometrial cells, the expression of PGE synthase is correlated with that of COX-2 and is an important enzyme for the production of PGE(2). Increasing this production will modulate the PGE(2)/PGF(2alpha) ratio and contribute to establishment of pregnancy.

The effects of estrogen and progesterone on prostaglandins and integrin beta 3 (beta3) subunit expression in primary cultures of bovine endometrial cells.

In cattle, endometrial expression of integrin alphavbeta3 is reduced on day 16 of the estrous cycle, coinciding with the critical period during which the decision is made to initiate luteolysis or continue with pregnancy. The objective of these experiments was to examine the relationship between estrogen and progesterone treatments, endometrial integrin alphavbeta3 expression, and prostaglandin F2alpha (PGF2alpha) and E2 (PGE2) production. Epithelial and stromal cells from intercaruncular (ICAR) and caruncular (CAR) bovine endometrium were treated with 17beta-estradiol (0.1 and 1.0 nM) and/or progesterone (1.0 and 10 nM) in a manner designed to mimic the steroid fluctuations of the estrous cycle. All cell types expressed estrogen receptor and progesterone receptor mRNA and protein. Intercaruncular stromal cells were the most responsive to steroidal regulation. Estrogen suppressed expression of integrin subunit beta3 mRNA in ICAR stromal cells (P< or =0.05). Progesterone and estrogen + progesterone treated cells did not differ in beta3 expression from controls (P> or =0.05). Steroid treatment did not affect PGF2alpha production in any cell type (P> or =0.05), however, estrogen decreased PGE2 production in all cells except CAR stroma (P< or =0.05). The results indicate that in bovine endometrium expression of integrin alphavbeta3 and production of PGE2 is influenced by estrogen.

Expression of the prostaglandin F synthase AKR1B1 and the prostaglandin transporter SLCO2A1 in human fetal membranes in relation to spontaneous term and preterm labor.

BACKGROUND: Human labor is a complex series of cellular and molecular events that occur at the materno-fetal and uterine levels. Many hypotheses have been proposed for the initiation of human labor, one hypothesis suggests that maturation of the fetus releases a signal in the amniotic fluid that will be transmitted to myometrium via the fetal membranes and initiate uterine contractions. There is strong evidence that prostaglandins (PGs) play a central role in initiation and progression of human labor. OBJECTIVES: In this study we intended to investigate the expression of prostaglandin F synthase and the prostaglandin transporter in the human fetal membranes and to explore the relationship between cytokines and PGs in the mechanism of human labor. METHODS: We used fetal membranes obtained before labor at term and after spontaneous labor at term or preterm to identify the changes in prostaglandin F synthase (AKR1B1) and human prostaglandin transporter (SLCO2A1) proteins in relation to parturition. Using fetal membranes explants we tested the effect of cytokines (interleukin-1 and tumor necrosis factor alpha) on PG production and the concomitant changes in cyclooxygenase-2 (PTGS2), AKR1B1 and SLCO2A1 expression. RESULTS: Expression of PTGS2 and AKR1B1 was upregulated in the fetal membranes in association with term labor while SLCO2A1 was downregulated with advancing gestation and during term labor. Before labor, IL-1 increased the expression of PTGS2, however during labor TNF upregulated PTGS2 and AKR1B1 proteins. CONCLUSIONS: The prostaglandin F synthase AKR1B1 is upregulated while prostaglandin transporter is downregulated during term labor. The amnion is more responsive than choriodecidua to stimulation with pro-inflammatory cytokines. The mechanisms of term and preterm labor are different.

Prostaglandin (PG) F2 alpha synthesis in human subcutaneous and omental adipose tissue: modulation by inflammatory cytokines and role of the human aldose reductase AKR1B1.

INTRODUCTION: PGF2α may be involved in the regulation of adipose tissue function. OBJECTIVES: 1) To examine PGF2α release by primary preadipocytes, mature adipocytes and whole tissue explants from the subcutaneous and omental fat compartments; 2) To assess which PGF synthase is the most relevant in human adipose tissue. METHODS: Fat samples were obtained by surgery in women. PGF2α release by preadipocytes, adipocytes and explants under stimulation by TNF-α, IL-1ß or both was measured. Messenger RNA expression levels of AKR1B1 and AKR1C3 were measured by RT-PCR in whole adipose tissue and cytokine-treated preadipocytes. The effect of AKR1B1 inhibitor ponalrestat on PGF2α synthesis was investigated. RESULTS: PGF2α release was significantly induced in response to cytokines compared to control in omental (p = 0.01) and to a lesser extent in subcutaneous preadipocytes (p = 0.02). Messenger RNA of COX-2 was significantly higher in omental compared to subcutaneous preadipocytes in response to combined TNF-α and IL-1ß (p = 0.01). Inflammatory cytokines increased AKR1B1 mRNA expression and protein levels (p≤0.05), but failed to increase expression levels of AKR1C3 in cultured preadipocytes. Accordingly, ponalrestat blunted PGF2α synthesis by preadipocytes in basal and stimulated conditions (p≤0.05). Women with the highest PGF2α release by omental adipocytes had a higher BMI (p = 0.05), waist circumference (p≤0.05) and HOMAir index (p≤0.005) as well as higher mRNA expression of AKR1B1 in omental (p<0.10) and subcutaneous (p≤0.05) adipose tissue compared to women with low omental adipocytes PGF2α release. Positive correlations were observed between mRNA expression of AKR1B1 in both compartments and BMI, waist circumference as well as HOMAir index (p≤0.05 for all). CONCLUSION: PGF2α release by omental mature adipocytes is increased in abdominally obese women. Moreover, COX-2 expression and PGF2α release is particularly responsive to inflammatory stimulation in omental preadipocytes. Yet, blockade of PGF synthase AKR1B1 inhibits most of the PGF2α release.

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions.

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1ß in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1ß is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1ß particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

The human aldose reductase AKR1B1 qualifies as the primary prostaglandin F synthase in the endometrium.

CONTEXT: Prostaglandins (PGs) E2 and PGF2α are produced in the endometrium and are important for menstruation and fertility. Dysmenorrhea is associated with increased production of PGF2α relative to PGE2, and the opposite is true for menorrhagia. The pathways leading to PGE2 biosynthesis are well described, but little is known for PGF2α. Aldoketoreductase (AKR)-1C3, the only PGF synthase identified in the human, cannot explain the production of PGF2α by endometrial cells. AKR1B1 appears to be an alternate candidate with promising therapeutic value. OBJECTIVE: The objective of the study was to address whether AKR1B1 (gene ID 231) is a functional PGF2α synthase in the human endometrium and a valid therapeutic target for menstrual pain. DESIGN: The design of the study was basic laboratory analyses to identify gene expression and protein levels associated with PGF2α production in endometrial tissues and endometrial cells from cycling women aged between 23 and 52 yr undergoing biopsies or hysterectomy for diverse gynecological disorders. RESULTS: AKR1B1 is expressed at a high level during the menstrual cycle during the secretory phase and in both epithelial and stromal cells, whereas AKR1C3 was found only in epithelial cells. Purified recombinant AKR1B1 protein, gene silencing, and transient transfection experiments all concur to demonstrate that this enzyme is a functional PGF synthase. Ponalrestat, a specific inhibitor developed to block AKR1B1 activity, reduced PGF2α production in response to IL-1ß in both cultured endometrial cells and endometrial explants. CONCLUSIONS: The human aldose reductase AKR1B1 currently associated with diabetes complications is also a highly functional PGF synthase responsible for PGF2α production in the human endometrium and a potential target for treatment of menstrual disorders.

The multidrug resistance-associated protein 4 (MRP4) appears as a functional carrier of prostaglandins regulated by oxytocin in the bovine endometrium.

Prostaglandins (PG) are involved in several female reproductive processes, and their action is regulated at the levels of biosynthesis, catabolism, and signal transduction. Facilitated transport across cell membranes emerges as an additional checkpoint regulating PG action. We have already reported on the influx transporter solute carrier organic anion transporting polypeptide (SLCO2A1) [PG transporter (PGT)] in relation to PG action in the bovine endometrium. In the present study, we report on the functional expression and regulation of multidrug resistance-associated protein 4 (MRP4)/ATP-binding cassette carrier 4, an alternate PG transporter belonging to the ATP-binding cassette carrier (ABC) family. We have found that MRP4 protein was present throughout the estrous cycle and exhibited a pattern of expression similar to that of PGT with maximal expression during early-mid luteal phase in the bovine endometrium. Functional expression and regulation of MRP4 was studied in vitro using the newly developed bovine endometrial epithelial bEEL and stromal CSC cell lines. Oxytocin (OT) stimulated PGF2α production and MRP4 mRNA and protein in a time- and dose-dependent manner but had no effect on PGT. OT induced preferred accumulation of PG outside the cells and secretion toward the basolateral side of polarized bEEL cells grown on membrane inserts. MK-571 and indomethacin, two documented inhibitors of MRP4 activity, blocked preferred accumulation of PG, but interferon-τ and NS-398 had no effect on MRP4 expression or the direction of PG transport. Our results suggest that MRP4 is a functional PG carrier under the regulation of OT in the bovine endometrium.

Epidermal growth factor receptor is an obligatory intermediate for oxytocin-induced cyclooxygenase 2 expression and prostaglandin F2 alpha production in bovine endometrial epithelial cells.

Oxytocin (OT) triggers the luteolytic pulses of prostaglandin F(2 alpha) (PGF(2 alpha)) from the endometrial epithelial cells in ruminants. We have proposed that the embryonic signal interferon-tau exerts its antiluteolytic effect by disrupting the OT signaling axis. Accordingly, we have attempted to define the signaling pathway of OT-induced PGF(2 alpha) production in the bovine endometrium using our newly characterized epithelial cell line (bEEL). OT receptor was coupled to the classical G alpha(q) pathway as evidenced by calcium release and activation of phospholipase C. Similarly, OT-induced PGF(2 alpha) production was mediated through the canonical ERK1/2 pathway. Because of the importance of receptor and nonreceptor tyrosine kinases in G protein-coupled receptor signaling, we studied the role of epidermal growth factor receptor (EGFR), c-Src, and phosphoinositide 3-kinase (PI3K) on OT-induced PGF(2 alpha) production in association with cyclooxygenase 2 (COX2) expression and ERK1/2 and Akt phosphorylation. The EGFR inhibitor AG1478 (10 microm) nearly abolished basal and OT-induced PGF(2 alpha) production and down-regulated COX2 expression and ERK1/2 phosphorylation. Because the transactivated EGFR can serve as a ligand for the signaling proteins with Src homology 2 (SH2) domain, we hypothesized a role for c-Src and PI3K in OT-induced PGF(2 alpha) production. Inhibitors of c-Src (PP2, 10 microm) and PI3K (LY294002, 25 microm) produced a significant decrease in OT-induced PGF(2 alpha) production and reduced COX2 expression. Also, PP2, but not LY294002, decreased OT-induced ERK1/2 phosphorylation. Because LY294002 did not affect ERK1/2 phosphorylation, but inhibited PGF(2 alpha) production and down-regulated COX2 expression, it is likely that the Akt pathway is also involved in PGF(2 alpha) production. Thus, EGFR may simultaneously activate c-Src and PI3K to amplify the OT signaling to increase the output of PGF(2 alpha) in bEEL cells.

Oxytocin receptor down-regulation is not necessary for reducing oxytocin-induced prostaglandin F(2alpha) accumulation by interferon-tau in a bovine endometrial epithelial cell line.

Interferon-tau (IFNtau) is the embryonic signal responsible for pregnancy recognition in ruminants. The primary action of IFNtau is believed to be mediated through inhibition of prostaglandin F(2alpha) (PGF(2alpha)) released from the endometrial epithelial cells in response to oxytocin (OT). Our working hypothesis was that the antiluteolytic effect of IFNtau also involved modulation of PG production downstream of OT receptor (OTR) and/or cyclooxygenase 2 (COX2). There is currently no OT-sensitive endometrial cell line to study the molecular mechanisms underlying our hypotheses. Therefore, we established an immortalized bovine endometrial epithelial cell line (bEEL) exhibiting OT response. These cells were cytokeratin positive, expressed steroid receptors, and exhibited preferential accumulation of PGF(2alpha) over PGE(2). The bEEL cells were highly sensitive to OT, showing time- and concentration-dependent increase in COX2 transcript and protein and PGF(2alpha) accumulation. Interestingly, IFNtau (20 ng/ml) significantly reduced OT-induced PGF(2alpha) accumulation, but surprisingly, the effect was not mediated through down-regulation of either OTR or COX2. Rather, IFNtau up-regulated COX2 in a time- and concentration-dependent manner while decreasing OT-induced PG accumulation. This suggests that COX2 is not a primary target for the antiluteolytic effect of IFNtau. Because IFNtau reduced OT-stimulated PGF(2alpha) accumulation within 3 h, the mechanism likely involves a direct interference at the level of the OT signaling or transcription in addition to the down-regulation of OTR observed in vivo. In summary, bEEL cells offer a unique in vitro model for investigating the cellular and molecular mechanisms underlying OT and IFNtau response in relation with luteolysis and recognition of pregnancy in the bovine.

15-Hydroxyprostaglandin dehydrogenase in the bovine endometrium during the oestrous cycle and early pregnancy.

Prostaglandins (PG) are primary regulators of reproductive function. In ruminants, the relative production of PGE2 and PGF2alpha determines the return to a new oestrous cycle or to the establishment of pregnancy in response to a viable embryo. PG action depends on biosynthesis, transport and interaction with their receptors, which are all expressed differentially during the oestrous cycle. PGs are, however, local mediators and thus the onsite degradation by enzymes such as 15-hydroxyprostaglandin dehydrogenase (HPGD), also known as 15-PGDH, is another factor to consider in the regulation of physiological action. Little information is available on PG catabolism in the endometrium during the oestrous cycle or early pregnancy. The purpose of this study was to clone the bovine 15-PGDH, produce the recombinant protein and generate a specific antibody to study its activity and its expression in the endometrium during the oestrous cycle. We have found that the bovine 15-PGDH is highly homologous to the rat and human isoforms. 15-PGDH is localized principally in the glandular epithelium and to a lesser extent in stromal and luminal epithelial cells. The enzyme expression is regulated during the oestrous cycle and it reaches its maximal level on days 16-18. Transient expression is observed in luminal epithelial and trophoblast cells on day 21 of pregnancy. The mRNA is expressed at a constant high level throughout the cycle. The activity of the recombinant 15-PGDH was also tested and was found comparable for PGF2alpha and PGE2. These data suggest that 15-PGDH contributes to the tight regulation of PG action in the endometrium especially at the critical period of recognition of pregnancy.

Decidualization and maintenance of a functional prostaglandin system in human endometrial cell lines following transformation with SV40 large T antigen.

Prostaglandins (PGs) are key regulators of reproductive function and associated pathologies. We have established stable endometrial stromal and epithelial cell lines with SV40 large T antigen (TAG) as a model to study PG action in the human endometrium. Two clones for each cell type were selected for rapid growth, PG production and response to interleukin-1beta (IL-1beta). The resulting stromal (HIESC) and epithelial (HIEEC) cells retain their characteristics for at least 40 population doublings (PDs). The selected clones express progesterone (PR) and estrogen receptor-alpha (ER-alpha) at both mRNA and protein levels. By contrast, with the existing known human endometrial cell lines Ishikawa and KLE, HIESC and HIEEC increase their production of PGF2alpha and PGE2 and cyclooxygenase (COX)-2 protein expression in response to IL-1beta. The latter cells also express the main biosynthetic enzymes involved in PG production, cytosolic phospholipase A2 (PLA2), COX-1 and COX-2, PGF synthase and PGE synthase and the corresponding EP2, EP3, EP4 and FP receptors. The selective COX-2 inhibitor NS-398 completely inhibits the increased production of PGs induced by IL-1beta in both cell types, whereas dexamethasone (DEX) exerts a stronger inhibition in HIESC than in HIEEC. The latter observation may be related to the higher expression of COX-1 measured in HIEEC. On the basis of the present characterization and previous determination of corresponding primary cell cultures, HIESC and HIEEC appear appropriate to study the contribution of PGs in the regulation of human endometrium function and associated pathologies.