A. Faletti

Interaction between uterine PGE and PGF2α production and the nitridergic system during embryonic implantation in the rat

Embryonic implantation is a complex process in which both maternal and embryonic signals are involved. In the present study, we evaluated changes in uterine prostaglandins production and nitric oxide synthase (NOS) activity during the course of early pregnancy and their interaction during implantation in rats. Uterine phospholipase A2 (PLA2) activity is increased on days 5 (day of ovoimplantation) and 6, compared to preimplantation days (3 and 4). This enhanced activity might be responsible for the observed increase in uterine PGE and PGF2 alpha production observed on day 5 of pregnancy, which induces endometrial vascular permeability and decidualization. When embryo access to the uterus is impaired, the increase of PG production is suppressed. During postimplantation, PGE levels return to preimplantation values, while PGF2 alpha decreased with respect to preimplantation values. Uterine NOS activity is also increased on day 4 and reaches a maximum on day 5, with a profile similar to PGE and PGF2 alpha. Dexamethasone administered in vivo decreased uterine NOS activity on day 4 of pregnancy but not on day 5, suggesting the presence of at least two types of NOS enzymes in the early days of pregnancy. A competitive inhibitor of NOS, L-NAME (600 and 1000 microM) induced a decrease in PGE and PGF2 alpha production in uterine tissue on day 5 of pregnancy. These results suggest the existence of a physiologically relevant nitridergic system which modulates prostaglandin production in the rat uterus during embryonic implantation.

Beta-endorphin inhibits prostaglandin synthesis in rat ovaries and blocks induced ovulation

The aim of the present study was to explore the action of exogenous beta-endorphin on the number of oocytes ovulated and on prostaglandin (PG) production in ovaries isolated from pregnant mares serum gonadotropin/human chorionic gonadotropin(PMSG/hCG)-primed immature rats. An intrabursal injection of the opioid (0.084 microgram) was given 4 hours after hCG and the number of oocytes within the oviducts on the following morning was reduced (P < 0.05). The same effect was also attained with an intraperitoneal (IP) injection (0.5 microgram). The time course of PG synthesis was quantified in ovaries of rats treated with an IP injection. Eight hours after hCG, prostaglandin content increased (P < 0.01) and remained high until 12 hours after hCG (P < 0.001). This increase was inhibited by the in vivo treatment with beta-endorphin. On isolated ovaries, beta-endorphin (10(-8) M) had a clear inhibitory action on prostaglandin production. beta-Endorphins effect on prostaglandin synthesis in the ovaries is of importance in the ovulatory process. The possible physiological role of beta-endorphin merits further investigation.

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.

Control of Salivary Secretion by Nitric Oxide and Its Role in Neuroimmunomodulation

Abstract: 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.

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.

Hyperandrogenism alters intraovarian parameters during early folliculogenesis in mice

This study aimed to investigate how hyperandrogenism affects early folliculogenesis. Hyperandrogenism was induced in prepuberal female BALB/c mice by daily s.c. injection of dehydroepiandrosterone (60 mg/kg body weight in 0.1 ml sesame oil) for 10 consecutive days. Although hyperandrogenism increased the growth rate of primary follicles, it also increased ovarian oxidative stress (evaluated by the increase in lipid peroxidation, the decrease in superoxide dismutase activity and the fact that glutathione content was not modified). By using the annexin V/cytometry assay it was found that the excess of androgens decreased viable ovarian cells and increased early apoptotic ones. The increased lipid peroxidation induced enhanced ovarian prostaglandin E production. In addition, hyperandrogenism increased the number of T lymphocytes that infiltrate ovarian tissue and modified their phenotype (decreased CD4+ or helper and increased the suppressor/cytotoxic CD8+). The excess of androgens decreased the ovarian expression of the long isoform of leptin receptor (Ob-Rb, the only isoform expressed in the ovarian tissue) when compared with controls. All these alterations increased serum concentrations of oestradiol, a pro-apoptotic agent. It is concluded that the excess of androgens impairs early follicular development by modulating some endocrine and immune parameters that are either directly or indirectly related to follicular atresia.

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

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.

Seasonal changes in ovarian steroid hormone concentrations in the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus)

Knowledge of armadillo reproductive physiology is essential for developing ex situ and in situ assisted reproductive techniques for propagating and/or controlling populations of these animals. The present study included assessment of fecal sex steroids by radioimmunoassay, determining reproductive status via monitoring ovarian activity (in the wild) and therefore reproductive status, in wild females of the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus) in the southern hemisphere. Plasma and fresh fecal progesterone concentrations were not significantly correlated in either species. However, in both species, there was a significant positive correlation between plasma progesterone and dry fecal progesterone concentrations (r = 0.82, P < 0.05 and r = 0.60, P < 0.05, respectively). Dry fecal progesterone and estradiol concentrations were measured in one captive C. villosus (average baseline progesterone and estradiol concentrations 28.72 ± 11.75 ng/g dry feces and 3.04 ± 0.80 ng/g dry feces, respectively) and one captive C. vellerosus (average baseline progesterone and estradiol concentrations 14.05 ± 3.03 ng/g dry feces and 3.46 ± 1.20 ng/g dry feces, respectively) to detect hormonal peaks over 1 y; these occurred from late fall to early summer. Feces from wild C. villosus and C. vellerosus were also collected over 1 y to determine progesterone peaks, which occurred in winter and spring in both species (with no peaks during the summer or fall). Accordingly, C. villosus and C. vellerosus had a seasonal reproductive pattern. The significant correlations between dry fecal and plasma progesterone concentrations validated this method for monitoring reproductive status in these species.

ALTERED PROSTANOID PRODUCTION BY CUMULUS-OOCYTE COMPLEXES IN A RAT MODEL OF NON-INSULIN-DEPENDENT DIABETES MELLITUS

Ovulation, oocyte maturation and PGE and PGF2 alpha production by oocyte-cumulus complexes were evaluated in rats with non-insulin-dependent diabetes induced by neonatal streptozotocin. Diabetic rats had normal estrous cycles, but ovulated a lower number of oocytes at estrus. When oocytes from control and diabetic rats obtained at proestrus were matured in vitro during 1, 2 or 4 hours (hr) of culture, differences were not found in the percent of germinal vesicle breakdown between both experimental groups. PGE and PGF2 alpha accumulation was higher in ovulated oocyte-cumulus complexes when compared to immature or in vitro-matured oocyte-cumulus complexes in both normal and diabetic rats. When control and diabetic rats are compared, more PGE and PGF2 alpha accumulation was observed in immature, in vitro-matured and in ovulated oocyte-cumulus complexes. A lower number of oocytes ovulated and increased oocyte-cumulus complexes prostaglandin production has been observed in this mildly diabetic experimental model. These abnormalities are similar to those previously found when 10 day embryos were evaluated in non-insulin-dependent diabetic rats.

Synthesis and release of prostaglandins D2 and E2 by rat uterine tissue throughout the sex cycle. Effects of 17-β-estradiol and progesterone

Abstract The synthesis and release of prostaglandins (PGs) D2 and E2 by rat uterine tissue was studied during the whole sex cycle. The PGs released into the bathing solution after 60 min of incubation were measured by specific radioimmunoassays. It was found that PGD2 released at diestrous was significantly higher than at proestrous and estrous. We also observed that PGE2 produced at diestrous was significantly higher than at proestrous and estrous, i.e. both PGs follow the same pattern of production throughout the sex cycle, but in all the cases the uterine strips released higher amounts of PGE2 than of PGD2. The influence of the sex hormones on PGD2 and PGE2 synthesis, was also studied. We observed that the treatment of ovariectomized rats with 17-β-estradiol decreased significantly the synthesis and release of PGD2 and PGE2. On the other hand, progesterone treatment did not modify the production of PGE2 but decreased significantly the synthesis of PGD2. In conclusion, in the present study we have found that PGD2 and PGE2 production varied similarly during the sex cycle and that 17-β-estradiol negatively regulates their synthesis. In addition, we have found that progesterone depressed only PGD2 synthesis without affecting PGE2 production.