Dexamethasone-induced intrauterine growth restriction modulates expression of placental vascular growth factors and fetal and placental growth.

Prenatal exposure to glucocorticoids (GC) is a central topic of interest in medicine since GCs are essential for the maturation of fetal organs and intrauterine growth. Synthetic glucocorticoids, which are used in obstetric practice, exert beneficial effects on the fetus, but have also been reported to lead to intrauterine growth retardation (IUGR). In this study, a model of growth restriction in mice was established through maternal administration of dexamethasone during late gestation. We hypothesised that GC overexposure may adversely affect placental angiogenesis and fetal and placental growth. Female BALB/c mice were randomly assigned to control or dexamethasone treatment, either left to give birth or euthanised on days 15, 16, 17 and 18 of gestation followed by collection of maternal and fetal tissue. The IUGR rate increased to 100% in the dexamethasone group (8 mg/kg body weight on gestational days 14 and 15) and pups had clinical features of symmetrical IUGR at birth. Dexamethasone administration significantly decreased maternal body weight gain and serum corticosterone levels. Moreover, prenatal dexamethasone treatment not only induced fetal growth retardation but also decreased placental weight. In IUGR placentas, VEGFA protein levels and mRNA expression of VEGF receptors were reduced and NOS activity was lower. Maternal dexamethasone administration also reduced placental expression of the GC receptor, αGR. We demonstrated that maternal dexamethasone administration causes fetal and placental growth restriction. Furthermore, we propose that the growth retardation induced by prenatal GC overexposure may be caused, at least partially, by an altered placental angiogenic profile.

Sperm Release From the Oviductal Epithelium Depends on Ca(2+) Influx Upon Activation of CB1 and TRPV1 by Anandamide.

The oviduct acts as a functional sperm reservoir in many mammalian species. Both binding and release of spermatozoa from the oviductal epithelium are mainly modulated by sperm capacitation. Several molecules from oviductal fluid are involved in the regulation of sperm function. Anandamide is a lipid mediator involved in reproductive physiology. Previously, we demonstrated that anandamide, through activation of the cannabinoid receptor type 1 (CB1), promotes sperm release from bovine oviductal epithelial cells, and through CB1 and the transient receptor potential vanilloid 1 (TRPV1), induces sperm capacitation. Herein we investigate co-activation between CB1 and TRPV1, and Ca(2+) influx as part of the mechanism of action of anandamide during sperm release from oviductal cells. Our results indicate that in the absence of Ca(2+) anandamide failed to release spermatozoa from oviductal epithelial cells. Additionally, sperm release promoted by cannabinoid and vanilloid agonists was abolished when the spermatozoa were preloaded with BAPTA-AM, a Ca(2+) chelator. We also determined Ca(2+) levels in spermatozoa preloaded with FURA2-AM co-cultured with oviductal cells and incubated with different cannabinoid and vanilloid agonists. The incubation with different agonists induced Ca(2+) influx, which was abolished by CB1 or TRPV1 antagonists. Our results also suggest that a phospholypase C (PLC) might mediate the activation of CB1 and TRPV1 in sperm release from the bovine oviduct. Therefore, our findings indicate that anandamide, through CB1 and TRPV1 activation, is involved in sperm release from the oviductal reservoir. An increase of sperm Ca(2+) levels and the PLC activation might be involved in anandamide signaling pathway.

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase.

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.

Fibronectin induces capacitation-associated events through the endocannabinoid system in bull sperm.

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.

Anandamide regulates lipopolysaccharide-induced nitric oxide synthesis and tissue damage in the murine uterus.

In women, the association between chronic marijuana smoking and early miscarriage has long been known. Anandamide, a major endocannabinoid, mimics some of the psychotropic, hypnotic and analgesic effects of Delta(9)-tetrahydrocannabinol, the psychoactive component of marijuana. The uterus contains the highest concentrations of anandamide yet discovered in mammalian tissues and this suggests that it might play a role in reproduction. The production of small amounts of nitric oxide (NO) regulates various physiological events including implantation and myometrial relaxation, but in an inflammatory setting such as sepsis, NO has toxic effects as it is a free radical. The results presented in this study indicate that anandamide modulates NO production induced by lipopolysaccharide (LPS) in an in-vitro murine model. It was shown that LPS-induced NO synthesis and tissue damage were mediated by anandamide, as a cannabinoid receptor type I antagonist could block the effect of LPS (P < 0.001). This endotoxin inhibited anandamide uterine degradation (P < 0.05) and increased the expression of one of its synthesizing enzymes (P < 0.05). Contrary to the known anti-inflammatory and protective effects, in this model anandamide seems to act as a pro-inflammatory molecule modulating the production of NO induced by LPS. This proinflammatory effect of anandamide may be implicated in pathological reproductive events such as septic abortion.

Dual effect of anandamide on rat placenta nitric oxide synthesis.

Anandamide (AEA) has been reported to have pleiotropic effects on reproduction, but the mechanism by which it exerts these effects is unclear. The aim of this study is to characterize rat placental endocannabinoid system and to analyze the possible functional role of AEA in the regulation of NO levels in rat placenta during pregnancy. We found that cannabinoids receptors (CB1 and CB2), FAAH and TRPV1 were expressed in chorio-allantoic placenta. NOS activity peaked at day 13 and decreased with progression of pregnancy. Both exogenous and endogenous AEA significantly decreased NOS activity. Although pre-incubation with AM251 (CB1 antagonist) or AM630 (CB2 antagonist) had no effect, co-incubation with both antagonists induced NOS activity. Furthermore, pre-incubation with exogenous AEA and both antagonists resulted in the induction of placental NOS activity and this effect was reverted with capsazepine (selective TRPV1 antagonist). Additionally, the enhanced NO synthesis caused by capsaicin was abrogated by co-treatment with capsazepine, illustrating that NOS activity could be modulated by TRPV1. Finally, the inhibition of TRPV1 receptor by capsazepine caused a significant fall in NOS activity. These data support the concept that AEA modulates NO levels by two independent pathways: (1) diminishing the NOS activity via CBs; and (2) stimulating NO synthesis via TRPV1. We hypothesized that AEA have an important implication in the normal function of placental tissues.

Nitric oxide (NO) inhibits prostaglandin E2 9-ketoreductase (9-KPR) activity in human fetal membranes.

Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E(2) 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE(2) into PGF(2alpha), the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2mM) increased PGE(2) production and diminished PGF(2alpha) synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [(3)H]-PGE(2) into [(3)H]-PGF(2alpha) and 13,14-dihidro-15-keto prostaglandin F(2alpha) (the PGF(2alpha) metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF(2alpha).

Expression analysis of cannabinoid receptors 1 and 2 in B cells during pregnancy and their role on cytokine production.

The endocannabinoid system consists in a family of lipids that binds to and activates cannabinoid receptors. There are two receptors so far described, the cannabinoid receptor 1 (CB1) and 2 (CB2). In the context of pregnancy, the endocannabinoid system was shown participates in different key aspects of reproductive events. B-lymphocytes are pleiotropic cells belonging to the adaptive arm of the immune system. Besides immunoglobulin production, B-lymphocytes were recently shown to be actively involved in antigen presentation as well as cytokine production, thus playing a central role in immunity. In this study we first aimed to characterize the expression of CB1 and CB2 receptors in B cells during pregnancy and then analyze the impact of their activation in term of cytokine production by B cells from pregnant and non-pregnant mice. We observed that the expression of CB1 and CB2 receptors in B-lymphocytes is differentially regulated during pregnancy. While CB2 expression is down regulated CB1 is augmented in B-lymphocytes of pregnant mice. Additionally, the treatment of activated B-lymphocytes with specific CB1 and CB2 agonists, showed a different response in term of cytokine production. Particularly, CB1 against boosted the production of the anti-inflammatory cytokine IL-10 by activated B-lymphocytes from pregnant mice.

Involvement of hypoxia and inflammation in early pregnancy loss mediated by Shiga toxin type 2.

INTRODUCTION: Symptomatic or asymptomatic Shiga toxin producing Escherichia coli (STEC) infections during early pregnancy may cause maternal or fetal damage mediated by Shiga toxin type 2 (Stx2). The aim of this study is to elucidate the mechanisms responsible for early pregnancy loss in rats treated with Stx2. METHODS: Sprague Dawley pregnant rats were intraperitoneally injected at day 8 of gestation with a sublethal dose (0.5 ng of Stx2/g of total body weight, 250 µl) of purified Stx2. Control rats were injected with the same volume of PBS. The expression of globotriaosylceramide (Gb3) glycosphingolipid receptor for Stx2 was evaluated by thin-layer chromatography (TLC). Regions of hypoxia in decidual tissue were determined by pimonidazole immunohistochemistry and vascular endothelial growth factor (VEGF) expression by Western blot and immunohistochemistry. Tumor necrosis factor-alpha (TNF-α) levels in serum and decidual tissue were evaluated by ELISA. Serum progesterone levels were determined by RIA. RESULTS: Decidual tissue from both, control and Stx2-treated rats showed similar expression of Gb3 receptor. Intrauterine growth restriction was observed in Stx2-treated rats, associated with hypoxia and an increase of decidual TNF-α levels. Decrease of serum progesterone levels and decidual VEGF expression were also demonstrated. DISCUSSION: Our findings indicate that Stx2 reaches the uteroplacental unit, binds Gb3 and triggers damage in decidual tissue. Poor oxygen supply accompanied with damage in the uteroplacental unit and inflammation could be responsible for the early pregnancy loss. Decrease in the pregnancy protective factors, serum progesterone and local VEGF, may contribute to the pregnancy loss.

The nitric oxide synthase of mouse spermatozoa.

Nitric oxide synthase (NOS) was evidenced in mature mouse spermatozoa by means of biochemical techniques and Western blot. During 120 min of incubation, 10(7) spermatozoa synthesized 7 +/- 2 pmol of L-[14C]citrulline. Besides, L-citrulline formation depended on the incubation time and on the concentration of L-arginine present in the incubation medium. Different concentrations of N(G)-nitro-L-arginine methyl ester (L-NAME) but not aminoguanidine, inhibited L-[14C]citrulline formation. Western-blot analysis of solubilized sperm proteins revealed a unique band of M(r)=140 kDa with the neural, endothelial and inducible NOS antisera tested. These results provide evidence that mature mouse sperm contains a NOS isoform and that spermatozoa have the potential ability to synthesize NO, suggesting a role for endogenous NO on mammalian sperm function.

Effects of cyclooxygenase inhibitor pretreatment on nitric oxide production, nNOS and iNOS expression in rat cerebellum.

1. The therapeutic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is thought to be due mainly to its inhibition of cyclooxygenase (COX) enzymes, but there is a growing body of research that now demonstrates a variety of NSAIDs effects on cellular signal transduction pathways other than those involving prostaglandins. 2. Nitric oxide (NO) as a free radical and an agent that gives rise to highly toxic oxidants (peroxynitrile, nitric dioxide, nitron ion), becomes a cause of neuronal damage and death in some brain lesions such as Parkinson and Alzheimer disease, and Huntington's chorea. 3. In the present study, the in vivo effect of three NSAIDs (lysine clonixinate (LC), indomethacine (INDO) and meloxicam (MELO)) on NO production and nitric oxide synthase expression in rat cerebellar slices was analysed. Rats were treated with (a) saline, (b) lipopolysaccharide (LPS) (5 mg kg(-1), i.p.), (c) saline in combination with different doses of NSAIDs and (d) LPS in combination with different doses of NSAIDs and then killed 6 h after treatment. 4. NO synthesis, evaluated by Bred and Snyder technique, was increased by LPS. This augmentation was inhibited by coadministration of the three NSAIDs assayed. None of the NSAIDs tested was able to modify control NO synthesis. 5. Expression of iNOS and neural NOS (nNOS) was detected by Western blotting in control and LPS-treated rats. LC and INDO, but not MELO, were able to inhibit the expression of these enzymes. 6. Therefore, reduction of iNOS and nNOS levels in cerebellum may explain, in part, the anti-inflammatory effect of these NSAIDs and may also have importance in the prevention of NO-mediated neuronal injury.

Role of nitric oxide in the metabolism of arachidonic acid in the rat anterior pituitary gland.

Nitric oxide (NO) affects cyclooxygenase (COX) and lipooxygenase (LOX) activities in several tissues. The aim of this study was to investigate the effect of NO on the AA metabolism in the anterior pituitary. LOX and COX products from anterior pituitaries of Wistar male rats were determined by [14C]-AA radioconversion method. Sodium nitroprusside (NP, 0.5 mM) and DETA NONOate (1 mM), NO donors, decreased 5-hydroxy-5,8,11,14-eicosatetraenoic acid (5-HETE) synthesis (P<0.05), effects that were reversed by hemoglobin. L-arginine also inhibited LOX activity. To the contrary, the inhibition of NO synthase by L-NAME (0.5 mM) or aminoguanidine (0.5 mM) increased 5-HETE production (P<0.05). COX activity was slightly stimulated by NP and L-arginine. However, DETA NONOate induced a stimulation of the synthesis of all prostanoids (P<0.05), this effect being reversed by hemoglobin. Neither NOS inhibitors nor hemoglobin modified basal prostanoids synthesis. These results indicate that NO inhibits LOX activity and stimulates COX activity in the anterior pituitary gland. The inhibition of LOX by NO may be another mechanism involved in the effects of NO on hormone release in the anterior pituitary.

Factors subserving the enhancing effect of progesterone on the output of prostaglandin F2 alpha in uteri from ovariectomized rats.

The effects of progesterone (P4) and of calcium-ionophore A-23187, on the release of prostaglandins (PGs) E2 and F2 alpha, in uteri isolated from ovariectomized rats and the influences of mepacrine and nifedipine, were explored. The metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF 1 alpha, PGE 2 and PGF2 alpha) in uterine segments from spayed rats, injected or not with P4, was also studied. In all cases ovariectomy was performed 20-25 days prior to sacrifice. One group of spayed rats were injected with 4.0 mg of P4 during two days and sacrificed 24 h after the last injection. The remaining spayed animals were considered as controls. Tissue samples from both groups were incubated for one hour in the absence or in the presence of either A-23187 (1.0 microgram/ml), mepacrine (10(-3) M) or nifedipine (10(-6) M), or a combination of A-23187 plus mepacrine. At the end of the incubating period PGs in the suspending solution were extracted, separated, identified (TLC) and quantitated. The metabolism of 14C-AA into different prostanoids was explored in uterine segments from spayed rats, injected or not with P4 prior to sacrifice. Tissue prepared from P4-injected rats as well as those from rats not receiving P4 but incubated with ionophore A-23187, generated and released significantly more PGF2 alpha into the incubating solution than basal controls, but failed to exhibit changes in the basal output of PGE.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between factors influencing Na(+)-K(+)-ATPase activity and eicosanoid production from 14C-arachidonic acid in spayed rat uterus.

Na(+)-K(+)-ATPase activity has been implicated in the metabolism of arachidonate and the release of prostaglandins (PG). The aim of the present study was to investigate a potential interaction between the activity of this enzyme and the production of bisenoic PG by uteri isolated from spayed rats. Ouabain or the incubation in low K+ medium (conditions which inhibit Na(+)-K(+)-ATPase) diminished the conversion of 14C-arachidonic acid to 6-keto-PGF1 alpha and PGF2 alpha and increased the production of TXB2. The incubation of uterine strips in a high K+ medium (condition which enhances Na(+)-K(+)-ATPase activity) increased the formation and release of 6-keto-PGF1 alpha and PGF2 alpha while the production of TXB2 and PGF2 diminished significantly. These observations suggested that the activity of Na(+)-K(+)-ATPase could modulate the production of PG and that could be involved in the alterations of the metabolism of eicosanoids found in several tissues during diabetes.

Influences of estradiol and of catechol and non-catechol estrogens on the output of prostaglandins in uteri from spayed rats.

The effects of 17-beta estradiol and of some catechol and non-catechol-estrogens on the synthesis and output of prostaglandins (PGs) E and F by uteri from ovariectomized rats, were explored. Uteri from castrated animals released twice as much PGE than PGF. When uterine tissue was obtained from spayed rats injected prior to sacrifice with a low dose of 17-beta estradiol (0.5 + 1.0 microgram, on two consecutive days), the output of PGE diminished significantly. With a higher dose of the hormone (0.5 + 50.0 micrograms) the depressive influence on the synthesis and release of PGE was even more marked, whereas the output of PGF rose significantly. Low or high doses of estrone or of estriol failed to affect the release of either one of the PGs determined. On the other hand, 2-0H-estradiol at a low dose had no action but at a higher one inhibited the release of PGE without influencing PGF. Neither low nor high doses of 2-0H estriol or of 2-0H estrone affected the synthesis and release of uterine PGs. It was also observed that all the compounds tested evoked a significant uterotrophic action. It appears plausible that some catechol metabolites of 17-beta estradiol, but not other catechol-estrogens, could be involved in the mechanism of action of 17-beta estradiol modulating the production of PGs by the rat uterus.

Mouse spermatozoa can synthesize PGE2 and 5-HETE in vitro: stimulatory action of nitric oxide.

Cauda epididymal spermatozoa were examined for their capacity to synthesize prostaglandins (PGs) and hydroxy acids and the role of nitric oxide (NO) on cyclooxygenase and lipoxygenase enzyme activity was determined. Sperm suspensions were incubated in the presence or absence of 300 microM sodium nitroprusside (NP) and 20 micrograms/ml hemoglobin (Hb) and then incubated with [14C]arachidonic acid (AA) for 60 min. The cyclooxygenase and lipoxygenase products of AA metabolism were measured for their radioactivity. It was found that mouse spermatozoa could synthesize PGE2 and 5-hydroxyeicosatetraenoic acid (5-HETE) under these experimental conditions. The synthesis of these two metabolites was 100% stimulated by NP, the releaser of NO, and the response to NP was completely prevented by Hb, a scavenger of NO. These data provide evidence that mouse spermatozoa can synthesize PGE2 and 5-HETE in the presence of AA in vitro and that NO is involved in the production of AA metabolites in the male gamete.

Role of interferon-alpha in arachidonic acid metabolism by the rat uteri from spayed animals: effect of 17 beta-estradiol.

Interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. We attempted to explore the effect of interferon (IFN)-alpha in the isolated rat uterus from ovariectomized animals on the metabolism of labelled arachidonic acid (AA). The experiments were performed with uterine preparations isolated from spayed rats treated or untreated with 17 beta-estradiol prior to sacrifice. The pre-incubation of uterine tissue isolated from ovariectomized rats with three different doses (4, 150 and 300 IU/ml) of IFN-alpha decreased the radioconversion of [14C]AA into 6-keto PGF1 alpha, PGF2 alpha and PGE2, whereas the formation of thromboxane B2 (TXB2) decreased after treatment with 4 IU/ml of INF-alpha but was augmented with 150 and 300 IU/ml. The three doses used diminished the synthesis of 5-hydroxyeicosatetraenoic acid (5-HETE) and increased the production of 12-HETE. We also studied the synthesis of uterine products of cyclooxygenase and lipoxygenase in uterine tissue from ovariectomized rats injected with 17 beta-estradiol and preincubated with IFN-alpha. We found that the three doses of IFN-alpha diminished the production of 6-keto-PGF1 alpha, PGE2, PGF2 alpha and TXB2 as well as 5-HETE and 12-HETE. These findings show that IFN-alpha modifies the basal synthesis of PGs from uterine tissue. Ovine and bovine trophoblastic protein prevents luteolysis by inhibiting the synthesis of PGF2 alpha. On the other hand, IFN-alpha shows 70% homology with ovine trophoblastic protein and binds to the same receptor. The results of the present study suggest that IFN-alpha may also act in the rat as a luteotrophic agent.

Role of nitric oxide on uterine and ovarian prostaglandin synthesis during luteolysis in the rat.

In previous studies in our laboratory, we demonstrated that oxytocin (oxy) augmented prostaglandin F(2alpha) (PGF(2alpha)) synthesis via enhancing the uptake of Ca2+ by uterine tissue. On the other hand, we have shown that oxy enhances PGF(2alpha) synthesis in uterine and ovarian tissues during the corpus luteum (CL) regression in the rat. In the present study we explore the possible relation between endogenous nitric oxide (NO) and oxy on PGs synthesis during the luteolytic phase in the rat. The experiments were done in uterine and ovarian preparations isolated from pseudopregnant (psp) rats during the luteolytic phase. Tissues were incubated "in vitro" with 1)- oxy (50 mU/ml), 2)-NMMA (N(G)-monomethyl-L-arginine), a potent NOs inhibitor (300 uM), and 3)- both reagents (oxy + NMMA). NMMA decreases the synthesis of both PGs (PGE and PGF(2alpha)) and oxy enhances PGF(2alpha) synthesis in uterine and ovarian tissue. When reagents were used in combination (oxy + NMMA), we found different results in uterus and ovaries; i.e., in uterine tissue the NO inhibition did not affect the increase of PGF(2alpha) synthesis by oxy. Meanwhile, in ovaries the oxy effect over the PGF(2alpha) synthesis was not seen when NOs was inhibited. Probably oxy acts via different mechanisms on PGF(2alpha) synthesis in uterine and ovarian tissue. This assumption was confirmed when the NOs activity in both tissues (uterine and ovarian) was measured after oxy treatment. We found that oxy enhanced the NOs activity in ovarian tissues from psp rats but did not modify the enzyme activity in uterine tissue.

Effect of an oxytocin receptor antagonist on ovarian and uterine synthesis and release of prostaglandin F2 alpha in pseudopregnant rats.

There is substantial experimental evidence suggesting that oxytocin has a role in luteolysis in ruminates. Endogenous pulses of uterine prostaglandin (PG) F2 alpha occur synchronously with pulses of oxytocin during luteolysis; leading us to propose a possible feedback loop between uterine PGF2 alpha and luteal oxytocin. In rates, the mechanism whereby oxytocin acts has not been well elucidated. In the present report, the effects of an oxytocin receptor antagonist in pseudopregnant rats were investigated. Pseudopregnancy was induced in immature female rats by gonadotrophin treatment; this resulted in the formation of corpus luteum that remained functional for 9 +/- 1 days. The pseudopregnant rats were assigned to one of the following four groups. In the first group the relationship between the release of ovarian and uterine PGF2 alpha was tested. We also studied the serum progesterone during the pseudopregnancy. We found that PGF2 alpha released into the incubation medium from ovaries of pseudopregnant rats increased (p < 0.05) and was maximal on day 9 of pseudopregnancy. This concentration remained high until day 10 of pseudopregnancy and then decreased. The PGF2 alpha released from the uterus to the incubation medium rose (p < 0.05) on day 8 of pseudopregnancy and reached the peak value on day 10. the serum progesterone was increased (p < 0.001) on day 2 pseudopregnancy and was greater on day 5 (p < 0.001). The second and third group received a specific oxytocin receptor antagonist (1-deamino-2-O-methyltyrosine) in two different concentrations (0.05 or 0.2 mumol/l before the peak of PG release. Both doses employed decreased (p < 0.001) the release into the incubating medium of PGF2 alpha from ovaries and uterus. Indeed, after the treatment, the progesterone levels were higher (p < 0.001) than control on day 10 of pseudopregnancy. In the fourth group, a potent inhibitor of cyclooxygenase activity was administered on day 8 of pseudopregnancy into the ovarian bursa. The serum progesterone levels increased (p < 0.01) compared to control suggesting a possible role of ovarian PG in the luteolytic phase of the corpus luteum regression. Thus, our findings show that oxytocin is luteolytic in pseudopregnant rats and this action is mediated by oxytocin receptors, as it was blocked by a specific oxytocin receptor antagonist.

High glucose levels modulate eicosanoid production in uterine and placental tissue from non-insulin-dependent diabetic rats during late pregnancy.

Severe uterine and placental disturbances have been described in diabetes pathology. The relative severity of these changes appears to correlate with high glucose levels in the plasma and incubating environment. In order to characterize changes in eicosanoid production we compared uterine and placental arachidonic acid conversion from control and non-insulin-dependent diabetes mellitus (NIDDM) rats on day 21 of pregnancy, into different prostanoids, namely PGE2, PGF22alpha, TXB2 (indicating the production of TXA2) and 6-keto-PGF1 (indicating the generation of PGI2). PGE2, PGF2alpha and TXB2 production was higher and 6-keto-PGF1alpha was similar in diabetic compared to control uteri. PLA2 activity was found diminished in the NIDDM uteri in comparison to control. A role for PLA2 diminution as a protective mechanism to avoid prostaglandin overproduction in uterine tissue from NIDDM rats is discussed. Placental tissues showed an increment in TXB2 generation and a decrease in 6-keto PGF1alpha level in diabetic rats when compared to control animals. Moreover, when control uterine tissue was incubated in the presence of elevated glucose concentrations (22 mM), similar generation of 6-keto PGF1alpha and elevated production of PGE2, PGF2alpha and TXB2 were found when compared to those incubated with glucose 11 mM. Placental TXB2 production was higher and 6-keto PGF1alpha was lower when control tissues were incubated in the presence of high glucose concentrations. However, high glucose was unable to modify uterine or placental prostanoid production in diabetic rats. We conclude that elevated glucose levels induced an abnormal prostanoid profile in control uteri and placenta, similar to those observed in non-insulin-dependent diabetic tissues.