It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The Alcohol suppresses reproductive function in humans, monkeys, and small rodents, such as the rat (1-4). In the rat,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3416 chronic administration of alcohol not only inhibits the estrous cycle but it can also delay onset of puberty (4, 5). In conscious animals, administration of alcohol via an indwelling gastric cannula in doses that produce mild intoxication inhibits pulsatile release of luteinizing hormone (LH), but not folliclestimulating hormone, within a few minutes (4). In this situation, the responsiveness of the pituitary gland to acute injection of LH-releasing hormone (LHRH) is unaffected, which shows that the mechanism of this effect is via suppression of the pulsatile LHRH release into the hypophyseal portal vessels that triggers release of LH from the gonadotropes of the adenohypophysis. The interference with estrous cycles and the delayed onset of puberty in the female rat is likely brought about by this suppression by ethanol of LHRH release (5). Secretion of LH is required to stimulate the ovary to produce ovarian steroids responsible for the estrous cycle, and the onset of puberty is consequently delayed.We have recently shown that nitric oxide (NO) controls the release of LHRH both in vivo and in vitro (6). On the basis of in vitro experiments employing incubation of medial basal hypothalamic (MBH) explants in a static incubation system, it has been determined that norepinephrine activates constitutive NO synthase (NOS) in neurons in this region (NOergic neurons) (6, 7). The NO released from these neurons diffuses to LHRH terminals, where it induces the release of LHRH, probably by activating cyclooxygenase 1. The activated cyclooxygenase then converts arachidonate into prostaglandin E2 (PGE2) (8). PGE2 activates adenylate cyclase, causing generation of cAMP, which acts via protein kinase A to evoke exocytosis of LHRH granules into the hypophyseal portal vessels for its delivery to the anterior pituitary gland (9, 10). The LHRH acts on the gonadotropes and causes a pulse of LH release. Support for this theoretical pathway stems from the ability of inhibitors of NOS, such as NG-monomethyl-Larginine, to inhibit LHRH release, whereas releasers of NO, such as sodium nitroprusside (NP), induce LHRH release both in vitro and in vivo (6-9).We have identified the site of inhibitory action of interleukin 1 (IL-1) on LHRH release...

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 mg 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.

ABSTRACT␤-Endorphin blocks release of luteinizing hormone (LH)-releasing hormone (LHRH) into the hypophyseal portal vessels by stimulating -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 ␤-endorphin exerts its inhibitory action on this NOergic pathway, medial basal hypothalami (MBH) from male rats were incubated with ␤-endorphin (10 ؊8 M). ␤-Endorphin decreased basal secretion of LHRH, and significantly inhibited the release of prostaglandin E 2 (PGE 2 ), a known stimulant of LHRH release. Incubation of MBH with ␤-endorphin at various concentrations (10 ؊9 -10 ؊6 M) in vitro decreased the activity of NO synthase (NOS) (measured by the conversion of [ 14 C]arginine to labeled citrulline). Conversely, the activity of NOS was increased by the -receptor antagonist, naltrexone (10 ؊8 M). Not only was the inhibitory action of ␤-endorphin on LHRH and PGE 2 release blocked by naltrexone (10 ؊8 M), but it increased NOS activity and LHRH and PGE 2 release. ␤-Endorphin also stimulated ␥-aminobutyric acid (GABA) release. Because GABA inhibits both nitroprusside (NPinduced PGE 2 and LHRH release by blocking the activation of cyclooxygenase by NO, this is another mechanism by which ␤-endorphin inhibits NP-induced PGE 2 and LHRH release. The results indicate that ␤-endorphin stimulates -opioid receptors on NOergic neurons to inhibit the activation and consequent synthesis of NOS in the MBH. ␤-Endorphin also blocks the action of NO on PGE 2 release and, consequently, on LHRH release, by stimulating GABAergic inhibitory input to LHRH terminals that blocks NO-induced activation of cyclooxygenase and consequent PGE 2 secretion.The endogenous opioid peptide, ␤-endorphin, plays an important role in inhibiting the release of luteinizing hormone (LH)-releasing hormone (LHRH) (1-3). Changes in release of ␤-endorphin within the hypothalamus are important in controlling the cyclic release of LHRH that induces the preovulatory surge of LH (3-5). Inhibitory opioid tone mediated by ␤-endorphin is removed in response to the increased plasma estrogen concentrations resulting from estrogen secretion by the preovulatory follicles, thereby facilitating the preovulatory release of LHRH. This tone maintains LHRH secretion at low levels during the rest of the estrous cycle of the rat and the menstrual cycle of monkeys (4). ␤-Endorphin acts mainly by activating -opioid receptors because -opioid receptor blockers such as naltrexone prevent its action (5).The mechanism by which ␤-endorphin inhibits LHRH release is not well understood. Recent evidence indicates that LHRH release is controlled primarily by nitricoxidergic (NOergic) neurons in the arcuate-median eminence region (6-9). The NO released from these neurons diffuses into the terminals of the LHRH neurons and activates not only guanylyl cyclase, leading to the release of cGMP, but also cyclooxygenase, leading to release of prostaglandins, i...

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.

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.

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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 dosedependent 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.

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