Differential effects of leptin on ovarian metalloproteinases and their tissue inhibitors between in vivo and in vitro studies.

In this study, we investigated the effect of leptin on the ovarian metalloproteinase system in the rat during the ovulatory process. Ovulation was induced in immature rats primed with gonadotropins. In both in vitro and in vivo experiments, we measured i) the protein expression of the ovarian metalloproteinases (matrix metalloproteinases, MMPs) and their tissue inhibitors (TIMPs) by western blot; ii) the gelatinase activity of the ovarian MMPs by zymography; and iii) the inhibitory action of TIMPs by reverse zymography. Using cultures of ovarian explants, leptin increased the activity but not the protein expression of MMP-2 and MMP-9 in both culture medium and ovarian tissue, and the protein expression of TIMPs, without a higher inhibitory action of the gelatinase activity. These results suggest either that the increase in TIMP proteins was not sufficient or that the inhibitory actions of TIMPs were impaired to suppress the MMP activity when the ovaries were directly exposed to leptin. To study the in vivo effect, rats received an acute treatment with high doses of leptin to inhibit ovulation. This treatment increased the expression of both the latent and the active forms of MMP-2 but did not result in a greater activity of MMP-2. In addition, the inhibitory action of TIMP-2 was also increased by this treatment. These results suggest that the administration of high doses of leptin could be regulating the follicle wall degradation, at least in part, by increasing the action of the ovarian TIMP-2 as a result of an extraovarian mechanism or signaling pathway.

Leptin modulates the expression of its receptors in the hypothalamic-pituitary-ovarian axis in a differential way.

To investigate the expression of leptin receptors (Ob-R) in the rat hypothalamus-pituitary-ovarian axis, immature rats were treated with eCG/hCG and Ob-R expression was evaluated by western blot analysis. The Ob-R expression increased 24 h after eCG administration in all the tissues assayed. In the hypothalamus, these levels immediately decreased to those obtained without treatment. In the pituitary, the Ob-R expression continued to be elevated 48 h after eCG administration, whereas the hCG injection did not modify these levels. Similar results were obtained with the ovarian long isoform. To assess the effect of leptin on its receptors, Ob-R was assessed in hypothalamus, pituitary and ovarian explants cultured in the presence or absence of leptin (0.3-500 ng/ml). In the hypothalamus, we found a biphasic effect: the Ob-R expression was either reduced or increased at low or high concentrations of leptin respectively. LH-releasing hormone secretion increased at 1 ng/ml. In the pituitary, Ob-R increased at 10 or 30 ng/ml of leptin for the long and short isoforms respectively. Leptin also induced an increase in LH release at 30 ng/ml. In the ovarian culture, the presence of leptin produced an increase in Ob-R expression at different ranges of concentrations and a dose-dependent biphasic effect on the progesterone production. In conclusion, all these results clearly suggest that leptin is able to modulate the expression of its own receptors in the reproductive axis in a differential way. Moreover, the positive or negative effect that leptin exerts on the ovulatory process may be dependent on this regulation.

Inhibitory effect of leptin on the rat ovary during the ovulatory process.

The aims of this study were to investigate the negative action of leptin on some intraovarian ovulatory mediators during the ovulatory process and to assess whether leptin is able to alter the expression of its ovarian receptors. Immature rats primed with gonadotrophins were used to induce ovulation. Serum leptin concentration was diminished 4 h after human chorionic gonadotrophin (hCG) administration, whereas the ovarian expression of leptin receptors, measured by western blot, was increased by the gonadotrophin treatment. Serum progesterone level, ovulation rate and ovarian prostaglandin E (PGE) content were reduced in rats primed with equine chorionic gonadotrophin (eCG)/hCG and treated with acute doses of leptin (five doses of 5 mug each). These inhibitory effects were confirmed by in vitro studies, where the presence of leptin reduced the concentrations of progesterone, PGE and nitrites in the media of both ovarian explants and preovulatory follicle cultures. We also investigated whether these negative effects were mediated by changes in the expression of the ovarian leptin receptors. Since leptin treatment did not alter the expression of ovarian leptin receptor, the inhibitory effect of leptin on the ovulatory process may not be mediated by changes in the expression of its receptors at ovarian level, at least at the concentrations assayed. In summary, the ovulatory process was significantly inhibited in response to an acute treatment with leptin, and this effect may be due, at least in part, to the direct or indirect impairment of some ovarian factors, such as prostaglandins and nitric oxide.

Leptin enhances ovulation and attenuates the effects produced by food restriction.

The aims of this study were to investigate the effects of chronic feed deprivation on the ovulatory process, and to assess whether leptin administration is able to alter these effects. Prepuberal rats subjected to food restriction and primed with gonadotrophins were used. Body and ovarian weights were significantly decreased in proportion to the severity of the food restriction. Only the most severe feed deprivation was able to inhibit the ovulation rate. Either buffer or leptin was daily administrated to prepuberal rats fed either ad libitum or with a severe food restriction. Serum progesterone, ovulation rate and ovarian prostaglandin E2 were reduced in rats subjected to food restriction and stimulated by daily administration of leptin in rats fed ad libitum. Negative effects produced by a severe food restriction were partially reversed by chronic administration of leptin. The ovarian endothelium nitric oxide synthase expression was strongly inhibited in rats with food restriction and once again, leptin administration reversed this effect. In summary, the ovulatory process was significantly inhibited in response to a severe decrease in food intake, at least in part, to the direct or indirect impairment of some ovarian factors production as prostaglandins and nitric oxide. Chronic treatment with leptin enhanced the ovulatory process in comparison with control animals, and partially prevented these negative effects produced by a severe malnutrition.

Interaction among beta-endorphin, nitric oxide and prostaglandins during ovulation in rats.

The aim of this study was to investigate the relationship between beta-endorphin and nitric oxide (NO) during the ovulatory process in rats. Immature rats were treated with equine chorionic gonadotrophin-hCG to induce ovulation. An intrabursal injection of beta-endorphin stimulated nitric oxide synthase (NOS) activity. This effect was completely reversed when naltrexone was co-injected with beta-endorphin. The stimulatory action of beta-endorphin on NOS activity was studied to determine whether it was exerted via prostaglandins. Treatment with prostaglandin E(2) (PGE(2)) completely reversed the beta-endorphin-induced stimulation of NOS activity. Moreover, intrabursal injection of meloxicam, an inhibitor of cyclooxygenase 2, increased NOS activity, but this effect was not altered by co-injection with beta-endorphin. The presence of both endothelial NOS (eNOS) and inducible NOS (iNOS) in the ovary at 10 h after hCG treatment was studied by western blot analysis. Local administration of beta-endorphin inhibited the expression of eNOS protein, whereas expression of iNOS protein was not detectable. Ovarian beta-endorphin content was diminished at 10 h after hCG injection. Treatment with prostaglandin synthesis inhibitors in vivo augmented the ovarian beta-endorphin content. In conclusion, these results indicate that beta-endorphin stimulates the activity of ovarian NOS indirectly by inhibiting prostaglandin production.

Inhibition of salivary secretion by lipopolysaccharide: possible role of prostaglandins.

Inducible (calcium-independent) nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are important in the regulation of the function of different organs during infection. A single dose of lipopolysaccharide (LPS; 5 mg/kg ip) within 6 h increased NOS activity (20%) and prostaglandin E (PGE) content (100%) in submandibular glands (SMG) and blocked stimulated salivary secretion in adult male rats. The administration of an iNOS synthesis inhibitor, aminoguanidine (AG), with LPS decreased NOS activity and PGE content. Furthermore, the administration of meloxicam (MLX), an inhibitor of COX-2, blocked the increase in PGE and the production of NO. The incubation of slices of SMG in the presence of 3-morpholinosydnonimine, a donor of NO, increased the release of PGE highly significantly. The incubation of SMG in the presence of a PGE(1) analog (alprostadil) increased the production of NO. These results indicate that LPS activates NOS, leading to NO release, which activates COX, generating PGEs that act back to further activate NOS, causing further generation of PGEs by activation of COX. Because the alprostadil administration inhibited stimulated salivation, LPS-induced inhibition of salivation appears to be caused by increased PGE production. Diminished salivary secretion produces poor oral health; thus the use of COX-2 inhibitors to counteract the effects of inhibited salivation should be considered.

Effects of aminoguanidine and meloxicam on nitric oxide and prostaglandin E production induced by lipopolysaccharide in the hypothalamus and anterior pituitary of the rat.

BACKGROUND/OBJECTIVE: Injection of bacterial lipopolysaccharide (LPS) into male rats activates genes that in turn induce many enzymes that participate in the animals' response to LPS. There is induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in many tissues. This induction could result from combination with cell surface LPS receptors that directly induce both genes, or the nitric oxide (NO) released as a result of iNOS induction could induce COX-2. METHODS: To distinguish between these two possibilities, specific inhibitors of iNOS and COX-2 activity, aminoguanidine (AG) and meloxicam (MLX), respectively, were injected either peripherally or intracerebroventricularly (i.c.v.), and their effect on NO and prostaglandin E (PGE) production induced by LPS in the medial basal hypothalamus (MBH) and anterior pituitary gland (AP) were determined. RESULTS: Peripheral injection of AG blocked iNOS-derived NO production in the AP but not in the MBH. When AG was injected i.c.v., iNOS-derived NO production in the MBH was blocked. MLX injected peripherally blocked COX-2-derived PGE(2) production in the MBH and AP, whereas AG injected peripherally or i.c.v. was ineffective. Since AG was only effective in blocking iNOS-derived NO production in the MBH when injected i.c.v., AG apparently does not effectively cross the blood brain barrier, whereas MLX injected peripherally inhibited PGE production, probably by inhibiting COX-2 activity in both the MBH and AP. AG was ineffective in preventing the increase in PGE derived from COX-2 in either the MBH or AP. CONCLUSION: LPS directly induces both enzymes, iNOS and COX-2, in the hypothalamus and AP.

High concentrations of inhibin A and inhibin B in ovarian serous cystadenoma: relationship with oestradiol and nitric oxide metabolites.

Inhibin production has been demonstrated in malignant epithelial ovarian tumours, but secretion of inhibins by benign cystadenoma has not yet been reported. The present study evaluated the concentrations of inhibin A and inhibin B and the relationship with oestradiol and nitric oxide metabolites in fluid collected from benign ovarian serous cystadenomas (n = 15). In addition, follicular fluid samples (n = 14) from women with regular ovulatory cycles undergoing ovarian stimulation for IVF were studied as a reference group. High concentrations of inhibin A (median = 89.3 ng/ml) and inhibin B (median = 116.1 ng/ml) were found in the cystic fluid of ovarian serous cystadenomas. These inhibin concentrations were even higher than in follicular fluid of stimulated follicles (inhibins A and B = 41.2 and 46.8 ng/ml respectively; P: < 0.001), whereas oestradiol was approximately 18-fold lower in cystic fluid than in follicular fluid (median = 34 versus 622 pg/ml, P: < 0.001). In ovarian cysts, the concentrations of inhibin A and oestradiol were inversely correlated (r = -0.678, P: = 0.008). Cystic fluid samples containing the highest concentrations of NO(2)(-)/NO(3)(-) (45-60 micromol/l) had lower inhibin A and higher oestradiol concentrations than those samples containing lower concentrations (10-25 micromol/l) of NO(2)(-)/NO(3)(-). It is concluded that high amounts of dimeric inhibins are present in ovarian serous cystadenoma. The source of inhibins and the determinants of the inverse association of inhibin A with oestradiol and nitric oxide remain to be determined.

Inhibitory pathways and the inhibition of luteinizing hormone-releasing hormone release by alcohol.

In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: gamma-aminobutyric acid (GABA) and beta-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10(-8) M), a micro-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by beta-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10(-5) M), naltroxone (10(-8) M), or both inhibitors added together. However, increasing the concentration of naltrexone (10(-6) M) but not bicuculline (10(-4) M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10(-5) M), naltroxone (10(-8) M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E(2), the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10(-4) M) but was blocked by naltroxone (10(-6) M), the action of alcohol can be accounted for by stimulation of beta-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release.

Control of salivary secretion by nitric oxide and its role in neuroimmunomodulation.

In many in vivo systems exposure to endotoxins (LPS) leads to the co-induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which is important to the regulation of the function of different systems during infection. In submandibular glands (SMG) neural (n)NOS is localized in neural terminals and in striated, granular convoluted and excretory ducts, endothelial (e)NOS in vascular endothelium and ducts, and iNOS in macrophages and in tubules and ducts. In normal adult male rats, injection of an inhibitor of NOS decreased the stimulated salivary secretion and a donor of NO potentiated it, indicating that NO exerts a stimulatory role. A single high dose of LPS (5 mg/kg, i.p.) induced an increase in NOS activity measured by the 14C-citrulline method, increased PGE content almost 100% as measured by RIA, and blocked stimulated salivary secretion. The administration of a specific iNOS inhibitor, aminoguanidine (AG), with LPS not only decreased NOS activity but significantly decreased PGE content, indicating that NO triggered the activation of COX-2. LPS increased conversion of labeled arachidonate to prostaglandins (PGs) showing that COX was induced. Since a PGE1 analogue blocked stimulated salivation, the LPS-induced inhibition of salivation is probably due to release of PGs. Therefore, the use of inhibitors of iNOS and COX-2 could be very useful to increase salivation during infection since saliva has antimicrobial actions.

Paracrine/autocrine control of female reproduction.

Neuropeptides, growth factors and cytokines are expressed in reproductive organs and tissues, where they interact with afferent endocrine messages to modulate cell proliferation and differentiation, local hormone secretion and vascular function. These events regulate complex processes such as gonadotropin pulsatility, ovulation, implantation and parturition. During reproductive life, a number of neuropeptides produced within the hypothalamus play a modulatory role in the control of gonadotropin-releasing hormone (GnRH) release, hence characterizing a hypothalamic paracrine system. The pituitary gland is a source and target of inhibin-related proteins, and these typical 'gonadal' products, once secreted by the pituitary cells, acquire the function of paracrine modulators of follicle-stimulating hormone (FSH) secretion. In the ovary, the effect of gonadotropins is locally modulated by growth factors acting in an autocrine/paracrine manner, although their precise role in folliculogenesis remains uncertain. Numerous local factors are involved in the control of endometrial growth, differentiation, receptivity and menstruation. Alterations in the paracrine endometrial system may underlie pathological processes such as infertility or endometrial neoplasia. The human placenta and its related membranes produce cytokines, hormones and growth factors that participate in the control of gestational development as well as in the maternal-fetal adaptation to gestational diseases. There is increasing evidence that paracrine signaling plays a fundamental role in all spheres of female reproductive function, and future research will concentrate on clarifying which of these local mechanisms play a decisive role in both physiology and disease, thus giving rise to new therapeutic strategies.

[Menstrual prostaglandin and dysmenorrhea: modulation by non-steroidal antiinflammatory drugs]. / Prostaglandinas menstruales y dismenorrea su modulación por antiinflamatorios no esteroideos.

The analgesic efficacy and tolerance of lysine clonixinate (LC) as well as LC-induced changes in menstrual prostaglandin levels were studied according to a prospective double-blind randomized crossover design, controlled with ibuprofen (I) and placebo (P). Treatment consisted in 4 consecutive phases: in the first phase, patients refrained from taking medication and during the remaining three phases, they received double-blind fixed doses of 1 tablet of lysine clonixinate 125 mg, I 400 mg or P, q.6 h. at random, three days before onset of menses and during 8 days thereafter. Controls were carried out at each menstrual cycle, assessing pain according to a scale from 0 to 4, onset of premenstrual and intramenstrual symptoms, relief of pain and occurrence of side-effects. During menstruation, patients recorded their assessments of pain in a diary and collected the whole menstrual bleeding during the first three days. The intensity of menstrual pain remained unchanged in controls upon admission (3.16) and during the phase with no treatment (3.04), but was significantly reduced with P (2.4), LC (1.79) and I (1.54). Significantly lower pain intensities compared with placebo were seen with active treatment phases. Forty-two percent of patients treated with P reported premenstrual pain which was significantly reduced to 17% with LC and to 12.5% with I. Active treatment phases revealed 21% of asymptomatic patients during premenstrual and menstrual periods and 71% (LC) and 75% (I) of cases with partial relief of pain. Patients' diaries showed significant pain reductions with LC and I, during the 1st and 2nd days compared with P; such differences were gradually reduced to nil by the 4th day. Levels of menstrual PGs changed according to pain intensity reductions from baseline (P: 29%, (NS); LC: 58% and I: 61%; both were statistically significant, p < 0.01).

Activity of ovarian nitric oxide synthase (NOs) during ovulatory process in the rat: relationship with prostaglandins (PGs) production.

Nitric oxide (NO) is synthesized by the rat ovary and a role in the follicular development, the ovulation, and the luteal formation has been postulated. The aims this study were to determine the activity of nitric oxide synthase (NOs) enzyme during the ovulatory process and to demonstrate the existence of a relationship between the ovarian NO production and the synthesis of prostaglandins (PGs) involved in the follicular rupture. Prepuberal rats treated with PMSG/hCG to induce ovulation were used. The NOs activity, measured by [(14)C]citrulline formation, showed an increase after PMSG administration and reached a maximum at 10 h after hCG injection. NOs activity remained high up to 24 h post ovulation. At 10 h after the hCG injection, the activity of Ca(2+)-dependent NOs (constitutive NOs) was similar to that seen at 0 h, and the activity of Ca(2+)-independent NOs (inducible NOs) increased from 14.4 to 51% of total activity. The in vitro ovarian production of PGE and PGF(2alpha) was inhibited by L-NAME and stimulated by 3-morpho-linosydnonimine (SIN-1), a NO donor. The in vivo production of ovarian prostaglandins was also inhibited by the intrabursal administration of two NOs inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine (L-NMMA). Our results suggest that the inducible NOs (iNOs) is the main isoform involved in the ovulatory process and that the NO produced stimulates the synthesis of both PGE and PGF(2alpha) from the cyclooxygenase pathway, to enhance the process of follicle rupture.

Inhibins: paracrine and endocrine effects in female reproductive function.

A great deal of new information has arisen in the past 2 years concerning the physiology of inhibins and their clinical relevance in reproductive medicine. It is now recognized that the two inhibin isoforms, inhibin A and inhibin B, are produced by the gonads in the course of gamete maturation and have different patterns of secretion during the menstrual cycle. Inhibins are also produced by the placenta and fetal membranes and may be involved in physiological adaptation of pregnancy. Clinically, inhibins may serve as sensitive tumor markers in postmenopausal women, or as useful tools for evaluating ovarian reserve in infertile women; they may also be used in the diagnosis of materno-fetal disorders.

beta-Endorphin blocks luteinizing hormone-releasing hormone release by inhibiting the nitricoxidergic pathway controlling its release.

beta-Endorphin blocks release of luteinizing hormone (LH)-releasing hormone (LHRH) into the hypophyseal portal vessels by stimulating mu-opiate receptors, thereby inhibiting secretion of LH. LHRH release is controlled by release of nitric oxide from nitricoxidergic (NOergic) neurons in the basal tuberal hypothalamus. To determine whether beta-endorphin exerts its inhibitory action on this NOergic pathway, medial basal hypothalami (MBH) from male rats were incubated with beta-endorphin (10(-8) M). beta-Endorphin decreased basal secretion of LHRH, and significantly inhibited the release of prostaglandin E2 (PGE2), a known stimulant of LHRH release. Incubation of MBH with beta-endorphin at various concentrations (10(-9)-10(-6) M) in vitro decreased the activity of NO synthase (NOS) (measured by the conversion of [14C]arginine to labeled citrulline). Conversely, the activity of NOS was increased by the mu-receptor antagonist, naltrexone (10(-8) M). Not only was the inhibitory action of beta-endorphin on LHRH and PGE2 release blocked by naltrexone (10(-8) M), but it increased NOS activity and LHRH and PGE2 release. beta-Endorphin also stimulated gamma-aminobutyric acid (GABA) release. Because GABA inhibits both nitroprusside (NP-induced PGE2 and LHRH release by blocking the activation of cyclooxygenase by NO, this is another mechanism by which beta-endorphin inhibits NP-induced PGE2 and LHRH release. The results indicate that beta-endorphin stimulates mu-opioid receptors on NOergic neurons to inhibit the activation and consequent synthesis of NOS in the MBH. beta-Endorphin also blocks the action of NO on PGE2 release and, consequently, on LHRH release, by stimulating GABAergic inhibitory input to LHRH terminals that blocks NO-induced activation of cyclooxygenase and consequent PGE2 secretion.

Prostaglandinas menstruales y dismenorrea su modulación por antiinflamatorios no esteroideos. / [Menstrual prostaglandin and dysmenorrhea: modulation by non-steroidal antiinflammatory drugs]

The analgesic efficacy and tolerance of lysine clonixinate (LC) as well as LC-induced changes in menstrual prostaglandin levels were studied according to a prospective double-blind randomized crossover design, controlled with ibuprofen (I) and placebo (P). Treatment consisted in 4 consecutive phases: in the first phase, patients refrained from taking medication and during the remaining three phases, they received double-blind fixed doses of 1 tablet of lysine clonixinate 125 mg, I 400 mg or P, q.6 h. at random, three days before onset of menses and during 8 days thereafter. Controls were carried out at each menstrual cycle, assessing pain according to a scale from 0 to 4, onset of premenstrual and intramenstrual symptoms, relief of pain and occurrence of side-effects. During menstruation, patients recorded their assessments of pain in a diary and collected the whole menstrual bleeding during the first three days. The intensity of menstrual pain remained unchanged in controls upon admission (3.16) and during the phase with no treatment (3.04), but was significantly reduced with P (2.4), LC (1.79) and I (1.54). Significantly lower pain intensities compared with placebo were seen with active treatment phases. Forty-two percent of patients treated with P reported premenstrual pain which was significantly reduced to 17

with LC and to 12.5

with I. Active treatment phases revealed 21

of asymptomatic patients during premenstrual and menstrual periods and 71

(LC) and 75

(I) of cases with partial relief of pain. Patients diaries showed significant pain reductions with LC and I, during the 1st and 2nd days compared with P; such differences were gradually reduced to nil by the 4th day. Levels of menstrual PGs changed according to pain intensity reductions from baseline (P: 29

, (NS); LC: 58

and I: 61

; both were statistically significant, p < 0.01).

Nitric oxide mediates increased prostaglandin E production by oocyte-cumulus complexes in the non-insulin-dependent diabetic rat.

Previous work described an increase in prostaglandin E (PGE) production by oocyte-cumulus complexes (OVA) obtained from non-insulin-dependent diabetic rats. More recently, it has been found that in control OVA nitric oxide (NO) mediates hCG-induced PGE secretion. To determine whether increases in PGE secretion by diabetic OVA are mediated by NO, the present study has evaluated the secretion of PGE by diabetic OVA, cultured in the absence or presence of hCG, NO donors (sodium nitroprusside (NP) and 3-morpholino-sydnonimine-hydrochloride (SIN-1)), and a NO synthase inhibitor (N(G)monomethyl-L-arginine; L-NMMA). hCG, NP and SIN-1 increased PGE secretion by diabetic OVA. L-NMMA did not modify basal secretion of PGE by control OVA but lowered PGE production in diabetic OVA to control values. L-NMMA prevented the hCG-induced PGE accumulation in control and diabetic OVA, and the quantities of PGE produced were similar to those of control OVA but lower than in diabetic OVA incubated in the absence of hCG. The effect of L-NMMA seems to be specific since N(G)monomethyl-D-arginine had no effect. NO synthase activity was higher in diabetic ovaries than in controls. The present results suggest that NO mediates the increased PGE production by diabetic OVA, probably a result of overproduction of NO.

Increased prostaglandin E generation and enhanced nitric oxide synthase activity in the non-insulin-dependent diabetic embryo during organogenesis.

Embryonic development, prostaglandin E (PGE) generation and nitric oxide synthase (NOS) activity during organogenesis were evaluated in an experimental rat model of non-insulin-dependent diabetes (NIDD) generated by neonatal administration of streptozotocin. Gross malformations were detected in 5% of NIDD embryos and these embryos were all non-viable; in the other 95%, growth was retarded but no congenital abnormalities were found. Control embryos were all alive and not malformed. The NIDD 11-day embryos secreted more PGE into the incubation medium than did controls. The NO donor SIN-1 increased PGE production in both control and NIDD embryos. A NOS inhibitor (L-NMMA) reduced PGE generation in both experimental groups, suggesting a modulatory role of NO on embryonic PGE production. Activity of NOS was higher in NIDD 11-day embryos than in controls. Treatment in vivo of control and NIDD rats (Days 7-11 of gestation) with a NOS inhibitor (L-NAME; 5 mg kg(-1) i.p.) reduced embryonic PGE production and induced a higher resorption rate and an increase in neural-tube defects. The results suggest that NO modulates PGE generation in the organogenetic embryo. In the NIDD model, overproduction of NO is observed, this NO probably enhancing embryonic PGE production. The relationship between PGE generation and the appearance of congenital abnormalities is discussed.

Uterine nitric oxide and prostaglandin E during embryonic implantation in non-insulin-dependent diabetic rats.

In the process of embryo implantation in the rat, both nitric oxide and prostaglandins act as vascular and myometrial regulators. The aim of the present work was to evaluate the effect of diabetes on the synthesis of both agents during embryo implantation. In diabetic rats, uterine activity of the enzyme nitric oxide synthase and prostaglandin E production were increased during peri-implantation compared to the control group (P < 0.05 and P < 0.001, respectively). Both parameters showed a prolonged increase in temporal profile during peri-implantation days. Local production of nitric oxide and prostaglandin E in the implantation sites was higher in diabetic rats (P < 0.05), but the intersite:site ratio was similar to that of the control group. On the other hand, the implantation rate and the timing of the beginning of this process were not altered in the diabetic group. These results suggest that the vasoactive modulators of the implantation process, nitric oxide and prostaglandins, are increased in this diabetic pathology, and that this increase is probably functioning as a compensatory mechanism, so as to allow an unaltered rate of embryo implantation in this model.

Naltrexone enhances ovulation and prostaglandin synthesis in the rat ovary.

We explored the action of beta-endorphin (beta E) and naltrexone (Nal) on the number of oocytes and on prostaglandins (PGE and PGF2 alpha) production by the ovaries from PMSG/hCG-primed immature and cycling rats. Superovulated rats were injected with beta-endorphin (0.5 microgram) intraperitoneally 4 hours after hCG. The number of ova ovulated was inhibited and this effect was blocked with naltrexone injected into the ovarian bursa (0.1 microgram) 30 minutes before beta-endorphin. Furthermore, beta-endorphin (10(-8) M) decreased prostaglandins production by ovaries isolated 4 hours after hCG. Intraperitoneal injection of beta-endorphin (0.5 microgram) at 17:00 hr on proestrus decreased (-23%) the number of ova within oviducts on the day after (estrus). Naltrexone injected intraperitoneally (5 micrograms) at 16:30 hr on proestrus increased the number of ova (+23%). On the other hand, beta-endorphin increased the number of oocytes obtained by puncture of antral follicles (+37%) and naltrexone decreased the number of oocytes (-33%). Prostaglandins content in the ovary of adult rats at 23:00 hr, approximately 4 hr before the onset of ovulation, was diminished when the rats received beta-endorphin at proestrus. Moreover, when the rats were injected with naltrexone, ovarian production of prostaglandins was increased. Our results further support the hypothesis that beta-endorphin affects ovulation at the level of the ovary in the rat and that endogenous opioids may be modulating this physiological process.