Eric Asselin

Cellular Mechanisms Involved during Oxytocin-Induced Prostaglandin F2α Production in Endometrial Epithelial Cells in Vitro: Role of Cyclooxygenase-21

PGs are important regulators of reproductive processes. At the time ofluteolysis in vivo, PGF2alpha is produced by endometrial cells, in response to oxytocin (OT). The mechanism by which OT induces the release of PGF2alpha remains to be defined. We have used 13 different cultures of bovine epithelial endometrial cells to study the effect of OT on the regulation of PGF2alpha and to identify the possible involvement of cyclooxygenases (COXs). OT induced a dose-dependent increase of both inositol phosphates (IPs) and [Ca2+]i concentration in epithelial cells labeled with [3H]-myoinositol or loaded with fura-2 (using a fluorescent microscope imaging system), respectively. OT induced a dose-dependent increase of both PGF2alpha production and COX-2 gene expression (as demonstrated by RT-PCR and Northern blots). PGF2alpha production was increased from 13.3 +/- 2.0 to 166.8 +/- 22.5 ng/ml (P < 0.0001). On the other hand, COX-2/beta-actin mRNA gene expression (as determined by densitometric analysis) was increased 5.1 +/- 0.7-fold (P < 0.001) with OT (10[-7] M) treatment, compared with control. Addition of indomethacin (1 microM) and a specific COX-2 inhibitor (NS-398, 1 microM) blocked the OT-induced PGF2alpha production. COX-1 and phospholipase A2 mRNA were expressed at steady-state levels, but no effect of OT was detected on their regulation. Combined to OT, 10 microq/ml of recombinant ovine interferon-tau (roIFN-tau) was able to decrease significantly (P < 0.0001) the dose-dependent increase of PGF2alpha production. Furthermore, partial bovine COX-1 (777 pb) and COX-2 (449 bp) cDNAs were cloned and sequenced. An homology of 83% and 97% was found in relation with rat and sheep, for COX-1, respectively. COX-2 was found to bear 84%, 86%, and 87% of homology in relation to rat, guinea pig, and human, respectively. Collectively, these results demonstrate, for the first time, that COX-2 is involved in the mechanism by which OT regulates PGF2alpha production in the endometrium.

IFN-τ increases PGE2 production and COX-2 gene expression in the bovine endometrium in vitro

Prostaglandins (PGs) are well known for their role in reproductive processes. At the time of pregnancy recognition, PGF2alpha is luteolytic and PGE2 may be antiluteolytic and luteotropic. During the preimplantation period, interferon-tau (IFN-tau) is produced by the conceptus and plays a crucial role in maternal recognition of pregnancy in domestic ruminants. We have demonstrated previously that recombinant bovine and ovine interferon-tau (rbIFN-tau and roIFN-tau) stimulate PGE2 production in epithelial cells, changing the primary PG produced by these cells from F2alpha to E2. In stromal cells, where PGE2 is the major PG produced, roIFN-tau induced an increase of both types of PGs. The aim of this paper is to identify the possible involvement of cyclooxygenases (COXs) in the modulation of PG production by trophoblastic interferons. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses (1, 10 and 20 microg/ml) of roIFN-tau. PG levels in the culture media were measured by enzyme immunoassays (EIA) and total RNA was extracted from the cells. Northern blot analysis was performed to quantify cyclooxygenase COX-1 (constitutive), COX-2 (inducible) and phospholipase A2 (PLA2) messenger RNA (mRNA) production in response to treatment. The results indicate that roIFN-tau treatment did not affect COX-1 and PLA2 mRNA production in either cell type, whereas COX-2 expression was upregulated in both. The up-regulation of COX-2 transcript was greater in stromal than in epithelial cells. The increase in COX-2 mRNA levels was concurrent with increased production of PGE2 and PGF2alpha in stromal cells and principally PGE2 in epithelial cells. Furthermore, addition of indomethacin (1 microM) and a specific COX-2 inhibitor (NS-398, 1 microM) blocked the roIFN-tau-stimulation of PG production in both cell types. The mechanism whereby elevated COX-2 expression results in a selective increase of PGE2 in epithelial cells remains to be elucidated. In stromal cells, an increase in COX-2 mRNA levels may explain increased PG production. The overall effect of roIFN-tau in the two cell types is a net increase in PGE2 output.

Detection and Regulation of the Messenger for a Putative Bovine Endometrial 9-Keto-Prostaglandin E2 Reductase: Effect of Oxytocin and Interferon-Tau

Abstract During reproductive processes, prostaglandin (PG) E2 (PGE2) and PGF2α play important roles in which they often exert opposite effects. At the time of recognition of pregnancy in vivo, PGF2α is recognized as the luteolytic factor in ruminants and in most species including human, whereas PGE2 may exert a luteoprotective action. We have previously demonstrated that recombinant interferon-tau (rIFN-τ), the embryonic signal responsible for recognition of pregnancy in ruminants, stimulated in vitro the production of PGE2 and prostaglandin-endoperoxide synthase 2 (Ptgs2; also called cyclooxygenase-2) gene expression in both epithelial and stromal endometrial cells. Since PGE2 is the major prostaglandin produced by stromal cells, the effect on Ptgs2 could explain the increase in PGE2 output. At high concentrations, however, recombinant ovine (ro) IFN-τ acts on epithelial cells by changing the primary PG produced from PGF2α to PGE2. This change in the primary PG produced could be explained by a decrease in PGF synthase (PGFS) activity or an increase in PGE synthase activity, or by modulation of a putative PGE2–9-ketoreductase, which converts PGE2 into PGF2α. Therefore, we have investigated the regulation of the mRNAs for PGFS and PGE2-9-ketoreductase (9K-PGR), two enzymes that lead to the production of PGF2α. Others have described 9K-PGR activity in uterus, ovaries, kidney, and liver of different species and have established that this enzyme could possess both 9K-PGR and 20α-hydroxysteroid dehydrogenase (20α-HSD) activity. Some have concluded that 9K-PGR and 20α-HSD are identical enzymes. Using primers sequences chosen from homologous nucleotide sequences of published rabbit 20α-HSD/9K-PGR and rat 20α-HSD cDNAs, a 317-base pair (bp) fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned, and sequenced. Homologies of 83% and 78% were found with rabbit and rat 20α-HSD, respectively. The presence of 20α-HSD/9K-PGR and prostaglandin F synthase (PGFS) mRNA expression was studied semiquantitatively in cultured epithelial cells using RT-PCR. Stimulation of cells with roIFN-t resulted in a biphasic response, an inhibition of PGF2α production at low dose (1 ng/ml) and a stimulation of PGE2 at high dose (10 μg/ml). The increase of PGE2 was accompanied by reduced 9K-PGR and PGFS mRNA gene expression. The effect of oxytocin (OT) was also studied, and the presence of OT had no effect on either 9K-PGR or PGFS gene expression. The 20α-HSD/9K-PGR transcript was also detected in other bovine tissues at different intensity (liver > kidney > testis > ovaries). We believe that the 9K-PGR and PGFS can be key enzymes in the regulation of specific PGs in the endometrium during the periimplantation period.

Interferon-Tau Stimulates Granulocyte-Macrophage Colony-Stimulating Factor Gene Expression in Bovine Lymphocytes and Endometrial Stromal Cells

Abstract Interferon-tau (IFN-τ), the antiluteolytic signal produced by the trophoblast prior to implantation in ruminants, exhibits immunomodulatory properties. It stimulates the production of prostaglandin (PG) E2 in bovine endometrial cells via the induction of cyclooxygenase-2 (COX-2). We previously demonstrated that preconditioning lymphocytes with PGE2 increases the expression of granulocyte–macrophage colony-stimulating factor (GM-CSF), a cytokine that promotes conceptus growth and survival. Our goal in the present study was to evaluate the impact of IFN-τ on the expression of GM-CSF in bovine peripheral blood lymphocytes (PBL) and endometrial epithelial and stromal cells. Changes in PGE2 production and mRNA levels of COX-2 were also studied in PBL in response to IFN-τ. Gene expression was estimated by semiquantitative reverse transcription–polymerase chain reaction and Northern analysis. The expression of GM-CSF in PBL was stimulated by treatment with IFN-τ. Furthermore, GM-CSF mRNA levels were increased after preconditioning PBL for 3 days with IFN-τ, followed by a 12-h restimulation without IFN-τ. Inhibition rather than stimulation of PGE2 production and COX-2 expression in PBL during treatment with IFN-τ suggests a direct effect on GM-CSF expression. Moreover, GM-CSF expression was stimulated in uterine stromal cells in response to IFN-τ. This study provides the first evidence for stimulation of GM-CSF expression by IFN-τ in both leukocytes and endometrial stromal cells. In view of the role of GM-CSF on fetal growth and survival, these results support the hypothesis that the conceptus mediates accommodation mechanisms in the uterus during early pregnancy by modulating the expression of beneficial cytokines at the fetomaternal interface.

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17β‐estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0‐1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose‐dependent manner (P < 0.01). Generation of inositol‐phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by 3H‐thymidine incorporation in serum‐free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose‐dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF2α production by epithelial cells (P < 0.0001) but had no effect on PGE2 production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro.

Prostaglandins are Primary Regulators of Conceptusmaternal Interactions Underlying Successful Pregnancy

Abstract Our laboratory is particularly interested in the characterization of conceptus-maternal interactions necessary for recognition, establishment, and maintenance of pregnancy. These interactions are evident very early during pregnancy and are necessary to ensure its successful outcome. The principal functions involved are the nutrition of the developing embryo, the maintenance of the corpus luteum the modulation of the immune system to prevent alto graft rejection, and regulation of myometrial contractility leading to rapid delivery of the mature fetus at term. Prostaglandins are key regulators of these functions. Prostaglandins are a class of locally acting factors having diffevent and even even opposite actions mediated by specific receptors. Clinically, prostaglandin E 2 (PGE 2 ), PGF 2α , and PGI 2 (prostacyclin) are particularly relevant to reproduction and obstetrics and gynaecology. Typical clinical disorders involving the metabolism of eicosanoids are: primary dysmenorrhoea, menorrhagia, pre-eclampsia, premature labour, and dystocia. We would like to present here the contribution of our research laboratory to the understanding of the role of prostaglandins in the regulation of conceptus-uterine interactions during pregnancy.