Regulation of Cyclooxygenase-2 and Prostaglandin F Synthase Gene Expression by Steroid Hormones and Interferon-τ in Bovine Endometrial Cells 1 (original) (raw)
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Reproductive Biology, 2007
Ovarian steroids modulate uterine receptivity in domestic species. Luteinizing hormone (LH) stimulates prostaglandin (PG)F 2α 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 LHstimulated PGF 2α 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 charcoalstripped 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 1 74 Steroids and LH modulate PGs secretion PGF 2α 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 2α (p<0.05) and PGE 2 (p<0.001) secretion. Moreover, LH increased PGF 2α 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 2α 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 cells responded to LH. Reproductive Biology 2007 7 (1):73-88.
Steroids, 2007
Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. The objective of this work was to characterize P4 metabolism by endometrial cells in vitro and determine if metabolites were able to modify prostaglandin secretion in response to oxytocin (OT). Endometrial epithelial and stromal cells were incubated with 3H-P4 or 3H-pregnenolone (P5) for 6 or 24 h. Metabolites in the medium were separated by HPLC. The results showed that P4 and P5 were converted to two major polar metabolites and a less polar metabolite that was identified as 5alpha- or 5beta-pregnanedione by LC/MS. Progesterone metabolism was similar in both stromal and epithelial cells. To determine if 5alpha- or 5beta-pregnanedione were able to modify PGF(2)alpha synthesis, cells were cultured with P4, 5alpha- or 5beta-pregnanedione (100 ng ml(-1)) for 48 h and then each group of cells was incubated for a further 4-6 h with or without OT (200 ng ml(-1)). Results showed that only P4 caused significant (P&amp;amp;amp;amp;amp;lt;0.001) increase in basal, but not OT-stimulated, PGF(2)alpha synthesis. OT binding assays showed no significant effect of progesterone or its metabolites on OTR concentration. In conclusion, bovine endometrial cells are able to metabolize pregnenolone and progesterone but neither 5alpha- nor 5beta-pregnanedione altered prostaglandin synthesis or OTR number in endometrial epithelial cells. These data suggest that 5-pregnanediones do not play a role in the regulation OT-stimulated PGF(2)alpha secretion during the bovine estrous cycle.
Ovarian steroids affect prostaglandin production in equine endometrial cells in vitro
Journal of Endocrinology, 2014
This study aimed to evaluate the influence of ovarian steroids on equine endometrial epithelial and stromal cells, specifically i) prostaglandin (PG) production in a time-dependent manner, ii) specific PG synthases mRNA transcription and protein expression, and iii) cell proliferation. After passage I, cells were exposed to vehicle, oxytocin (OT, positive control, 10 K7 M), progesterone (P 4 , 10 K7 M), 17b estradiol (E 2 , 10 K9 M), or P 4 CE 2 for 12, 24, 48, or 72 h. Following treatment, PG concentration was determined using the direct enzyme immunoassay (EIA) method. Alterations in PG synthases mRNA transcriptions, PG synthases protein expression, and cell proliferation in response to the treatments were determined after 24 h using real-time PCR, western blot, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide respectively. After 24 h, E 2 and P 4 CE 2 increased PGE 2 and PGF 2a secretion as well as specific prostaglandinendoperoxide synthase-2 (PTGS2), PGE 2 synthases (PGES), and PGF 2a synthases (PGFS) expression in the epithelial cells (P!0.05). Additionally, E 2 and P 4 CE 2 increased PTGS2 expression in stromal cells after 24 h (P!0.05). In stromal cells, P 4 CE 2 increased PGE 2 production as well as PGES expression after 24 h (P!0.05). Both E 2 and P 4 CE 2 increased PGF 2a production by stromal cells after 24 h (P!0.05). Ovarian steroids affected proliferation of stromal and epithelial cells during the 24-h incubation period (P!0.05). We provide evidence that ovarian steroids affect PG production in equine endometrial cells, upregulating PTGS2, PGES, and PGFS expression. Ovarian steroid-stimulated PG production could be an important mechanism occurring in the equine endometrium that is involved in the regulation of the estrous cycle and early pregnancy. Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 264 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 265 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 266 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 267 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 268 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 269 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 270 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 271 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 272 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 274 Research A Z SZÓ STEK and others Ovarian steroids on PG production in the mare 220:3 275
Biology of Reproduction, 1999
Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F 2␣ from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF 2␣ secretion by interferon-(IFN-) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-(rbIFN-) on OTinduced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT؉100 ng/ml rbIFN-for 3, 6, 12, and 24 h. OT significantly increased PGF 2␣ and PGE 2 secretion at all time points (p Ͻ 0.01), while rbIFN-inhibited the OTinduced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p Ͻ 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-suppressed the induction of COX-2 mRNA (89%, p Ͻ 0.01) and COX-2 protein (50%, p Ͻ 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-(p Ͻ 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-. The results showed that rbIFN-also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFNinhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.
Biology of Reproduction, 2009
11Beta-hydroxysteroid dehydrogenase (HSD11B) enzymes have important roles in regulating cortisol availability in target tissues. We previously demonstrated that HSD11B1 is expressed and active in bovine endometrium and that cortisol suppresses prostaglandin (PG) F2alpha and PGE2 production in cultured bovine endometrial stromal cells. The present study was conducted to examine whether locally synthesized PGF2alpha and/or PGE2 regulates the enzymatic bioactivity and/or the expression of HSD11B1 in bovine endometrium. The conversion rate of cortisone to cortisol in cultured endometrial stromal cells was significantly stimulated by PGF2alpha (1 and 10 lM). In a dose-dependent manner, PGF2alpha but not PGE2 increased the net conversion of cortisone to cortisol in stromal cells after 4 h of treatment. In addition, the bioactivity of HSD11B1 was significantly inhibited by indomethacin (10 lM). The inhibitory effect of indomethacin on HSD11B1 bioactivity was abolished by PGF2alpha (1 lM) but not by PGE2. Although PGF2alpha (1 lM) did not affect the expression of HSD11B1 mRNA in cultured stromal cells, it significantly stimulated the protein expression of HSD11B1. Cycloheximide, a general translational inhibitor, abolished the stimulatory effects of PGF2alpha on HSD11B1 protein expression in endometrial stromal cells, indicating that PGF2alpha increases HSD11B1 protein expression by stimulating a posttranscriptional process rather than a transcriptional mechanism. These results demonstrate that PGF2alpha but not PGE2 increases HSD11B1 bioactivity and protein expression by stimulating a posttranscriptional mechanism in stromal cells and suggest that cortisol has a physiologically relevant role in preventing excessive uterine PG production in nonpregnant bovine endometrium.
Biology of Reproduction, 1998
Prostaglandins (PGs) are important mediators regulating uterine functions during the reproductive process. The objective of this study was to examine, in myocytes from the circular and longitudinal layers of bovine myometrium, the relative levels of mRNA and proteins corresponding to the gene expression of key enzymes (phospholipase A 2 ; prostaglandin G/H synthase-1 [PGHS-1]; prostaglandin G/H synthase-2 [PGHS-2]; prostaglandin I 2 synthase) involved in PG biosynthesis. We examined the influence of estradiol-17 and progesterone on the expression and activity of these enzymes. Treatment of myocytes with progesterone (P 4 : 10 nM, 24 h) in the absence or presence of estradiol-17 (E 2 : 1 nM, 72 h) suppressed PG biosynthesis by approximately 60% in both myometrial layers. No significant effect was observed after E 2 treatment. The combined effect of E 2 and P 4 on PG accumulation was correlated with the modulation of PGHS-2 protein and mRNA levels in the two myometrial layers without affecting other enzymes of the PG cascade. Selective or nonselective inhibition of PGHS activity with CGP 28238 (PGHS-2-specific; a product from Ciba-Geigy: 6-[2,4-difluorophenoxy]-5-methyl-sulfonylamino-1-indanone) or indomethacin (PGHS-1 and-2) reduced prostacyclin accumulation (measured as 6-keto-PGF 1␣ in the culture medium) in a dose-dependent manner in the two myometrial layers. A significant inhibitory effect was obtained at a low concentration of indomethacin (1 nM, p Ͻ 0.05) compared to CGP 28238 (10 nM, p Ͻ 0.05). In both myometrial layers, the maximal effect of indomethacin and/or CGP 28238 on PG accumulation was observed at 100 nM and represented 85% and 65% inhibition, respectively. In the presence of phorbol 12-myristate (100 nM), CGP 28238 (10 nM) significantly suppressed PGHS-2 mRNA level by 44.80 ؎ 7.67% (p Ͻ 0.01) and 27.83 ؎ 7.62% (p Ͻ 0.05) in the longitudinal and circular layer, respectively. In contrast, indomethacin did not have any significant effect. These data constitute the first quantitative analysis of key enzymes involved in PG biosynthesis in separated myometrial layers. Furthermore, the results provide interesting information on the CGP 28238 drug modulating both enzymatic activity and mRNA expression of PGHS-2.
2005
In cattle, luteolysis results from the pulsatile release of endometrial prostaglandin F2α (PGF2α). Estradiol-17β (E 2 ) and progesterone (P 4 ) are involved in the regulation of luteolysis, but their mechanisms of action are unclear. The overall objective of this experiment was to investigate the actions of E 2 and P 4 on the control of PGF2α synthesis. Crossbred beef cows were slaughtered on Day 17 of a synchronized estrous cycle. Endometrial explants were incubated for 1 h with culture medium alone and for additional 11 h in culture medium supplemented with either 0, 10 -12 , or 10 -11 M E 2
Biology of Reproduction, 2003
Tumor necrosis factor-␣ (TNF␣) has been shown to be a potent stimulator of prostaglandin (PG) F 2␣ synthesis in bovine endometrial stromal cells. The aims of the present study were to determine the effect of interferon-(IFN) on TNF␣-stimulated PGF 2␣ synthesis and the intracellular mechanisms of TNF␣ and IFN action in the stromal cells. When cultured bovine stromal cells were exposed to TNF␣ (0.006-0.6 nM) for 24 h, the production of PGF 2␣ and cyclooxygenase (COX)-2 gene expression were stimulated by TNF␣ (0.06-0.6 nM, P Ͻ 0.05). Moreover, a specific COX-2 inhibitor (NS-398; 5 nM) blocked the stimulatory effect of TNF␣ on PGF 2␣ production (P Ͻ 0.05). Although IFN (0.03-30 ng/ml) did not stimulate basal PGF 2␣ production in the stromal cells, it suppressed TNF␣ action in PGF 2␣ production dose dependently (P Ͻ 0.05). Moreover, the stimulatory effect of TNF␣ (0.6 nM) on COX-2 gene expression was completely blocked by IFN (30 ng/ml; P Ͻ 0.05), although the gene expression of COX-2 was not influenced by IFN. The overall results indicate that the stimulatory effect of TNF␣ on PGF 2␣ production is mediated by the up-regulation of COX-2 gene expression and suggest that one of the mechanisms of the inhibitory effect of IFN on luteolysis is the inhibition of TNF␣ action in PGF 2␣ production in the stromal cells by the down-regulation of COX-2 gene expression stimulated by TNF␣.
Endocrinology, 2009
Before implantation, the porcine endometrium and trophoblast synthesize elevated amounts of luteoprotective prostaglandin estradiol-17 (E 2 ) (PGE 2 ). We hypothesized that embryo signal, E 2 , and PGE 2 modulate expression of key enzymes in PG synthesis: PG-endoperoxide synthase-2 (PTGS2), microsomal PGE synthase (mPGES-1), PGF synthase (PGFS), and PG 9-ketoreductase (CBR1) as well as PGE 2 receptor (PTGER2 and -4) expression and signaling within the endometrium. We determined the site of action of PGE 2 in endometrium during the estrous cycle and pregnancy. Endometrial tissue explants obtained from gilts (n ϭ 6) on d 11-12 of the estrous cycle were treated with vehicle (control), PGE 2 (100 nM), E 2 (1-100 nM), or phorbol 12-myristate 13-acetate (100 nM, positive control). E 2 increased PGE 2 secretion through elevating expression of mPGES-1 mRNA and PTGS2 and mPGES-1 protein in endometrial explants. By contrast, E 2 decreased PGFS and CBR1 protein expression. E 2 also stimulated PTGER2 but not PTGER4 protein content. PGE 2 enhanced mPGES-1 and PTGER2 mRNA as well as PTGS2, mPGES-1, and PTGER2 protein expression. PGE 2 had no effect on PGFS, CBR1, and PTGER4 expression and PGF 2␣ release. Treatment of endometrial tissue with PGE 2 increased cAMP production. Cotreatment with PTGER2 antagonist (AH6809) but not PTGER4 antagonist (GW 627368X) inhibited significantly PGE 2 -mediated cAMP production. PTGER2 protein was localized in luminal and glandular epithelium and blood vessels of endometrium and was significantly up-regulated on d 11-12 of pregnancy. Our results suggest that E 2 prevents luteolysis through enzymatic modification of PG synthesis and that E 2 , PGE 2 , and endometrial PTGER2 are involved in a PGE 2 positive feedback loop in porcine endometrium.
Animal Reproduction Science, 2019
Cyclooxygenase-2 (COX-2) has important functions in the synthesis and release of endometrial prostaglandin F2α (PGF2α). Excessive production of COX-2 leads to an increase in endometrial PGF2α synthesis and subsequently causes luteolysis and early embryonic mortality. The aim of this study was to investigate in goats the effects of COX-2 small interference RNA (siRNA) on COX-2 mRNA abundance and the secretion of PGF2α and PGE2 in goat endometrial cells. Endometrial cells isolated from goat uteri were cultured at 38.5°C and 5% CO 2. The cells were treated with different concentrations (0, 10, 25, 50, 100, 250, 500, 750 and 1000 nM per well) of three different COX-2 siRNAs at confluency for 24 h. At 24 h post culture, COX-2 mRNA abundance was quantified using qPCR and PGF2α and PGE2 concentrations were quantified in the culture medium. There was a lesser relative abundance of COX-2 mRNA in endometrial cells at 100 to 1000 nM siRNA. The greatest extent of abundance suppression, however, was observed with 1000 nM siRNA. Transfection of COX-2 siRNA (1000 nM) to endometrial cells suppressed the COX-2 mRNA abundance by 77%, 82%, and 84% with siRNA 1, 2, 3, respectively. Furthermore, with COX-2 siRNA transfected cells, there was a lesser (P < 0.05) PGF2α concentration than in cells not transfected, whereas PGE 2 secretion was not affected. The results of the study provide evidence that COX-2 siRNA used in this study suppresses COX-2 mRNA abundance and PGF2α secretion but there was no association between PGE2 concentrations and COX-2 mRNA abundance in goat endometrial epithelial cells.