Correlation of increased concentration of ovine endometrial cyclooxygenase 2 with the increase in PGE2 and PGD2 in the late luteal phase.

Ovine endometrium showed transient expression of high concentrations of the inducible isoform of cyclooxygenase, cyclooxygenase 2 (COX-2), whereas the constitutive isoform, cyclooxygenase 1 (COX-1), was expressed at much lower concentrations and did not change. In this study, the pattern of prostaglandin synthesis in endometrial luminal cells was investigated in relation to their COX-2 content. Endometrial cells from cyclic or pregnant ewes at days 9, 12, 14 and 16 were isolated and analysed for the presence of COX-1 and COX-2 proteins using western blot analysis. Freshly isolated cells were incubated with 0.5 microCi [3H]arachidonic acid ml-1. Radioactive cyclooxygenase metabolites were analysed by reverse-phase HPLC. Luminal cells produced mainly PGF2 alpha, PGE2, PGD2 and 13,14-dihydro-15-keto PGF2 alpha and to a lesser extent 6-keto PGF1 alpha, thromboxane B2 and 13,14-dihydro-15-keto PGE2. The production of PGE2 and PGD2 was proportional to the cellular concentration of COX-2. PGE2 and PGD2 release was low on day 9 when COX-2 was not expressed, whereas high concentrations of PGE2 and PGD2 were synthesized on days 12-14 when COX-2 was highly expressed, reaching 100 ng microgram-1 cellular protein. In contrast, the basal production of PGF2 alpha did not appear to be related to COX-2 concentration and was greatest on day 16. Moreover, the release of PGF2 alpha was maintained at steady state values between days 9 and 14 by the production of 13,14-dihydro-15-keto PGF2 alpha. Although PGF2 alpha output was lower at day 16 of pregnancy compared with the oestrous cycle, no difference was observed in the pattern of prostaglandin synthesis between pregnant and non-pregnant ewes.

Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period.

In this study we investigated expression of the two isoforms of the prostaglandin-forming enzyme, cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2), in sheep embryos. Using Western blot and immunohistochemical analyses, we demonstrated that Cox-2 was highly expressed in embryos from Day 8 to Day 17 of development whereas Cox-1 was undetectable during this time. The expression of Cox-2 was developmentally regulated. It was maximal between Days 14 and 16. There was a 30-fold increase in Cox-2 content per protein extract between Day 10 and Day 14, corresponding to a 50,000-fold increase in the whole embryo. The expression of Cox-2 declined after Day 16 to become undetectable by Day 25 of pregnancy. Cox-2 was localized in the trophoblastic cells and was not detected in the inner cell mass. The [3H]arachidonic acid metabolites synthesized by Cox-2-rich conceptuses were analyzed by HPLC after short-term embryo culture. Day 14 conceptuses released mainly cyclooxygenase metabolites and to a lesser extent lipoxygenase derivatives. Cyclooxygenase products were 6-keto-prostaglandin (PGF)1alpha 18.2% (+/- 4.2), thromboxane-B2 22.51% (+/- 15.9), PGF2alpha 21% (+/- 11), PGE2 14.5% (+/- 7.4), and PGD2 2.7% (+/- 2.6). Taken together, these results suggest an important role for the Cox-2-dependent cyclooxygenase metabolites during embryo development.

Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy.

In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.

Preferential esterification of arachidonic acid into ethanolamine phospholipids in epithelial cells from ovine endometrium.

In sheep, the pulsatile release of prostaglandin F2 alpha by the endometrium is necessary to achieve luteolysis which occurs at the end of the oestrous cycle. The production of prostaglandins is known to depend upon the availability of arachidonic acid, the fatty acid precursor of prostaglandin biosynthesis. Consequently, the mechanisms controlling intracellular amounts of arachidonate may be involved in the regulation of prostaglandin synthesis. Since arachidonic acid is mostly found in phospholipids and the endometrial epithelium is the primary source of prostaglandin F2 alpha during luteolysis, the fate of arachidonic acid when incorporated into epithelial cells from the ovine uterus was investigated. Endometrial epithelial cells isolated from cyclic ewes at day 15 after oestrus were cultured in the presence of [3H]arachidonic acid. Incorporation and distribution of the radiolabelled arachidonic acid into the various phospholipid classes were examined using HPLC. We observed that ethanolamine glycerophospholipids contained 61% of the total tritiated arachidonic acid incorporated into cellular lipids, whereas phosphatidylinositols, phosphatidylcholines and phosphatidylserines contained 17%, 13% and 4.7%, respectively. In addition, the radioactivity measured within phosphatidylethanolamines was preferentially detected in the 1-alkenyl-2-acyl (44%) forms of ethanolamine phospholipids, also called plasmalogens. The kinetic study of arachidonic acid uptake into ethanolamine phospholipids showed that arachidonic acid was rapidly esterified into the diacyl forms and then uptake decreased, whereas the incorporation increased continuously into the plasmalogen forms for at least 24 h. These results demonstrate that the primary pool of esterified arachidonic acid is found in ethanolamine plasmalogens of epithelial cells from the ovine endometrium. The high arachidonate content of ethanolamine plasmalogens suggests that these phospholipids play a crucial role in the control of arachidonic acid availability and ultimately in the regulation of prostaglandin synthesis.

Phospholipase A2 activity in endometrium from early pregnant and non-pregnant ewes.

In the ewe, synthesis of the luteolytic factor, prostaglandin F2 alpha, increases from day 13 to the end of the estrous cycle. Availability of free arachidonic acid is usually the rate-limiting step in prostaglandin biosynthesis. Phospholipase A2 (PLA2) may be the key enzyme for the hydrolysis of arachidonic acid from membrane-bound phospholipids. To investigate uterine PLA2 activity during the estrous cycle and early pregnancy, we monitored the release of [14C]oleic acid from the substrate 1-palmitoyl-2-[14C]oleoyl-phosphorylcholine by homogenates and cytosolic fractions of endometrium from ewes on days 12, 14 and 16 of the estrous cycle or pregnancy. We observed that PLA2 activity dropped by 58% (p < 0.02) in day-16 pregnant endometrium compared to day-16 non-pregnant endometrium. We then investigated whether the reduced PLA2 activity was due to induction of a specific inhibitor. The PLA2-inhibitor activity was determined by monitoring the inhibition of release of [14C]oleic acid from the radioactive substrate by porcine pancreatic PLA2. Inhibition by endometrial homogenates of pregnant animals of the control enzyme activity was 27% and only 14% by cyclic ones. Inhibition was dose-dependent and was as high as 53% (p < 0.01) with 1 mg protein from pregnant endometrial homogenates. Endometrial PLA2 behaved as a Michaëlian enzyme in the endometrium of day-16 cyclic ewes (Km = 79.4 mumol/l). Furthermore, the inhibitory activity from pregnant endometrium had characteristics of competitive inhibition. Our results suggest that inhibition of endometrial PLA2 activity could occur in early pregnant ewes.