Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.     

Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F_2α (PGF_2α), resulted in a very rapid increase in the intracellular levels of [³H]-inositol triphosphate (IP₃), [³H]-inositol biphosphate (IP₂), and [³H]-inositol monophosphate (IP₁) in cells prelabeled with [³H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2?2 min and declined to control levels by 6–10 min. The stimulation was dose-dependent with maximal increases observed near 10^?6 M PGF_2α. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP₃. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP₃ or on the accumulation of IP₁. In contrast, vanadate at 10^?6 or 10^?5 M did lead to a decrease in the breakdown of IP₁ and a concomitant increase in IP₁, IP₂, and IP₃. PGF_2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E₁ (PGE₁). PGE₁, in a dose-dependent manner, was found to inhibit the observed IP₃ increase by PGF_2α at 1 1/2 min of stimulation. PGF_2α treated and control cultures did not differ in cAMP or cGMP levels, cellular ⁴⁵Ca uptake, nor cellular ²²Na uptake. We propose that IP₃ may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF_2α and that it may play a crucial role with cAMP in growth regulation.     

IL-1 beta and prostaglandins regulate integrin mRNA expression.

The purpose of this study was to examine the effects of IL-1 beta on integrin expression in MG-63 human osteosarcoma cells. Human recombinant IL-1 beta (rIL-1 beta) produced significant increases in both alpha 2- and alpha 5-subunit mRNA levels, as well as a smaller increase in alpha v-subunit mRNA. In contrast, IL-1 beta decreased alpha 4-subunit mRNA levels by approximately 30% relative to untreated controls. These findings suggest that human IL-1 beta differentially regulates expression of integrins. When cultures were treated with both IL-1 beta and the cyclooxygenase inhibitor, indomethacin, the expression of alpha 2-, alpha 5-, and alpha v-subunit mRNA levels were dramatically increased relative to untreated controls; co-treatment with 0.5 mM prostaglandin E2 (PGE2) partially reversed this effect. Indomethacin alone did not affect integrin mRNA levels. Treatment with IL-1 beta or IL-1 beta + indomethacin also induced significant changes in MG-63 morphology (i.e., increased cell elongation) and increased the ability of cells to contract collagen gels. PGE2 reversed the above effects on cell morphology and gel contraction. These findings indicate that (a) IL-1 beta differentially regulates the expression of integrins and (b) that PGE2, which is induced by IL-1 beta, may provide a negative feedback loop which counteracts the stimulatory effect of IL-1 beta on integrin gene expression. It is suggested that products of inflammation may affect cell behavior by differentially regulating the expression of various integrins.     

Biosynthesis of prostaglandins in ovarian follicles and corpus luteum of sheep ovary

The biosynthetic potential of prostaglandins (PGs) was measured in ovarian follicles and corpus luteum of sheep ovary. The total prostaglandins formed under non-enzymatic conditions were much lower in comparison to that formed using native GSTs. When the GSTs of ovarian follicles were employed, the major prostaglandin formed was PGE2 (81.22%) followed by PGD2 (16.9%) and PGF2 alpha (1.87%). In case of corpus luteum, prostaglandin formed was PGF2 alpha (59.01%). Since PGF2 alpha was demonstrated to be the luteolytic factor, the present study indicates the formation of luteolytic factor in the ovarian tissue itself.     

Yin-yang: balancing act of prostaglandins with opposing functions to regulate inflammation

For many years, cyclooxygenase-2 (COX-2), a critical enzyme for PG production, has been the favorite target for anti-inflammatory drug development. However, recent revelations regarding the adverse effects of selective COX-2 inhibitors have stimulated intense debate. Interestingly, in the early phase of inflammation, COX-2 facilitates inflammatory PG production while in the late phase it has anti-inflammatory effects. Moreover, although some PGs are proinflammatory, others have anti-inflammatory effects. Thus, it is likely that PGs with opposing effects maintain homeostasis, although the molecular mechanism(s) remains unclear. We report here that an inflammatory PG, PGD2, via its receptor, mediates the activation of NF-kappaB stimulating COX-2 gene expression. Most interestingly, an anti-inflammatory PG (PGA1) suppresses NF-kappaB activation and inhibits COX-2 gene expression. We propose that while pro- and anti-inflammatory PGs counteract each other to maintain homeostasis, selective COX-2 inhibitors may disrupt this balance, thereby resulting in reported adverse effects.     

Fibroblastic regulation of osteoblast function by prostaglandins

The effects of osteogenic inhibitory factors secreted by human periodontal ligament fibroblasts were studied in rat bone marrow stromal cell cultures. Serum-free conditioned medium from cultures of fibroblasts strongly depressed formation of mineralized tissue by bone marrow cell cultures. The inhibitory activity was reduced by treatment of fibroblast cultures with indomethacin or by pretreatment of conditioned medium with specific antibodies to prostaglandins (PGs) E₂ and F₂. Passage of conditioned medium over octadecyl columns enriched PGs four-fold and significantly increased inhibitory activity. Inhibition of mineralization was replicated by treatment of bone-cell cultures with PGs B₂, D₂, E₂, and I₂ at concentrations of 350 ng/ml to 350 pg/ml. All combinations of these agents were inhibitory but PGE₂ and PGF₂ exhibited the greatest inhibition at low concentrations (350 pg/ml). These experiments indicate that fibroblasts secrete PGs which can inhibit bone formation, and this may be one mechanism whereby fibroblasts can modulate osteogenesis at the interfaces of soft and mineralizing connective tissues.     

Effect of prostaglandins on hypothalamic-pituitary-testicular function in vitro.

The effect of prostaglandins (PG) A1, E1, E2 and F2 alpha in the concentration range of 10(-7)--10(-4) M were studied in vitro on a rat hypothalamic tissue, collagenase-digested isolated anterior pituitary cell and Leydig cell suspension system by measuring the testosterone production of incubated Leydig cells. PGs did not change the testosterone production and the hCG sensitivity of the Leydig cells, nor the LH secretion and the LHRH sensitivity of the anterior pituitary cells. PGE2 at concentrations of 10(-6), 10(-5) and 10(-4) M significantly increased the hypothalamic tissue-induced pituitary-testicular activation, and this stimulatory effect of PGE2 was dose dependent. PGA1, PGE1 and PGF2 alpha did not alter hypothalamic LHRH release measured in vitro. The results suggest that PGE2 has a direct stimulatory effect on hypothalamic LHRH release.     

Endogenous prostaglandins regulate spontaneous contractile activity of uterine strips isolated from non-pregnant pigs

Myometrial strips isolated from non-pregnant pigs show spontaneous contractile activity. In the present study, the involvement of endogenous prostaglandins in regulation of uterine spontaneous contraction was investigated using mechanical, immunohistochemical and biochemical approaches. Immunohistochemical study and Western blot analysis for immunoreactive cyclooxygenase (COX) indicated that COX-1 but not COX-2 was expressed predominantly in the myometrium of non-pregnant pigs in a muscle layer-dependent manner (longitudinal muscle>circular muscle). Pretreatment of uterine strips with indomethacin and selective COX-1 inhibitors (SC-560 and FR122047) significantly reduced both the amplitude and frequency of spontaneous contraction in the longitudinal muscle, but inhibition by COX inhibitors was negligible in the circular muscle. On the other hand, CAY10404, a COX-2 inhibitor, did not change the spontaneous contraction in either of the muscle layers. Pretreatment with SC-560 reduced myometrial PGF(2alpha) and PGE(2) levels. Contractile FP and EP(3) receptors were expressed in a muscle layer-dependent manner (longitudinal muscle>circular muscle), similar to the expression pattern of COX-1. In conclusion, endogenous prostaglandins produced by COX-1 regulate spontaneous contractile activity of non-pregnant porcine uterine longitudinal muscle selectively due to the heterogeneous expression of contractile prostanoid receptors and COX-1.     

Function curve of the membranes that regulate amniotic fluid volume in sheep

Seven singleton 120-day fetal lambs were prepared with a shunt from the lung to the gastric end of the esophagus, a bladder catheter, and multiple amniotic fluid and vascular catheters. The urachus was ligated. Beginning 7 days later, amniotic fluid volumes were determined by drainage, followed by replacement with 1 liter of lactated Ringer (LR) solution. Urine flow into the amnion was measured continuously. In 14 of 27 experiments, amniotic fluid volumes were determined again 2 days after the inflow into the amnion had consisted of urine only and in 13 experiments after the inflow of urine had been supplemented by an intraamniotic infusion of LR solution. Intramembranous absorption was calculated from the inflows and the changes in volume between the beginning and end of each experiment. The relations between absorption rate and amniotic fluid volume, the "function curves," were highly individual. Urine production during the infusion of LR solution did not decrease, fetal plasma renin activity decreased (P < 0.001), and amniotic fluid volume increased by 140% [SE (27%), P < 0.005], but the increase in the amniochorionic absorption rate of 411% [SE (48%), P < 0.001] was greater (P < 0.005) than the increase in volume. Each of the seven fetuses was proven capable of an average intramembranous absorption rate that exceeded 4.5 liters of amniotic fluid per day. During the infusion of LR solution, the increase in the rate of absorption matched the rate of infusion (both in ml/h), with a regression coefficient of 0.75 (P < 0.001). Thus, even for large amniotic fluid volumes, volume is not limited by the absorptive capacity of the amniochorion, and, at least in these preparations, the position of the function curve and not the natural rate of inflow was the major determinant of resting amniotic fluid volume.     

Effect of bisenoic prostaglandins on the uterine vasculature of the nonpregnant sheep

The effects of the bisenoic prostaglandins on the uterine vasculature and uterine contractile activity have been evaluated in an unanesthetized chronically catheterized nonpregnant sheep preparation. Changes in uterine blood flow were monitored with electromagnetic flow probes while uterine contractile activity and tone were determined via an intra-uterine balloon connected to a pressure transducer. Prostaglandins A₂, D₂, E₂, and prostacyclin (PGI₂) were all found to be vasodilators. PGD₂ and PGI₂ were much more potent than PGA₂ and PGE₂ in dilating the uterine vasculature. The prostacyclin breakdown product 6-keto PGF_1α, PGF_2α, thromboxane B₂, and the endoperoxide analogues U44069 and U46619 produced vasoconstriction of the uterine vasculature. Prostaglandins A₂, D₂ and F_2α increased while PGI₂ decreased uterine contractile activity. PGF_2α also increased uterine tone suggesting that a portion of its vasoconstrictor activity may be due to mechanical compression of the uterine vasculature.     

Secretion of prostaglandins and leukotrienes by endometrial cells in cows with subclinical and clinical endometritis

The aims of this study were (1) to measure the secretion of prostaglandin F_2α (PGF_2α), prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄), and leukotriene C₄ (LTC₄) by endometrial cells collected by a cytobrush from healthy cows and cows with subclinical and clinical endometritis in the fourth week postpartum, and (2) to evaluate the relationship between the mediators' levels of secretion and the number of polymorphonuclear neutrophils (PMNs) in the uterine smears of cows with subclinical endometritis. The study included cows without any signs of clinical endometritis (n = 63) and cows with clinical endometritis as a positive control (ENDOM, n = 12). Two different threshold ratios (>5% and >18% of PMNs) were used to categorize the cows without clinical signs as with or without cytologic endometritis (CE). Considering the first or second threshold, the animals with CE were included in group CE POS I or CE POS II, whereas the healthy cows were assigned to group CE NEG I or CE NEG II, respectively.     

Evidence for involvement of prostaglandins in central alpha adrenergic activity and LH release in sheep

Circhoral pulsatile release of immunoreactive luteinising hormone (LH) and prostaglandin F2 alpha (PGF2 alpha) occur synchronously into the jugular vein in ovariectomised sheep. Following a 4-hour control period, intra-carotid injections of phentolamine or intramuscular injections of phenoxybenzamine were given to ovariectomised sheep and the pulsatile release of LH and PGF2 alpha was monitored for a further 6 to 8 hours. Phenoxybenzamine caused a fall in LH and PGF2 alpha in jugular venous plasma. Phentolamine also reduced LH significantly but in this case a marked rise in PGF2 alpha as measured by radioimmunoassay (RIA) occurred after very high doses of phentolamine. Interpretation of the latter results was complicated by the fact that phentolamine at high dose levels interfered with the RIA of PGF2 alpha in plasma. Experiments were repeated in ovariectomised sheep with cannulae placed in the lateral ventricles of the brain for sampling cerebrospinal fluid (CSF). In contrast to the previously observed rise in jugular venous PGF2 alpha following high doses of phentolamine, a fall in CSF levels of immunoreactive PGF2 alpha occurred following intracarotid phentolamine or phenoxybenzamine in 3 out of 7 experiments, while no change was observed in the remaining 4 animals. Phentolamine did not reduce LH significantly in animals with intraventricular cannulae. The work provides support for the view that circhoral pulses of immunoreactive PGF2 alpha in sheep are neural in origin and may be related to sympathetic neurotransmission.