Effects of long-term testosterone, gonadotropin-releasing hormone agonist, and pimozide treatments on gonadotropin II levels and ovarian development in juvenile female striped bass (Morone saxatilis)

The ability of the juvenile female reproductive axis to respond to hormonal stimulation was investigated in a Perciform fish, the striped bass (Morone saxatilis) using various combinations of testosterone (T), GnRH agonist (GnRHa), and pimozide. A long-term treatment with T alone, or T in combination with GnRHa, increased pituitary gonadotropin II (GtH II) levels 2- and 3-fold, respectively, suggesting that T and GnRHa each stimulate GtH II accumulation. Release of the accumulated GtH II could be induced only by high doses of GnRHa in combination with T, indicating that GtH II synthesis and release require different levels of GnRH stimulation. The addition of the dopamine antagonist pimozide did not affect pituitary and plasma GtH II levels but, in response to an additional acute GnRHa challenge, inhibited the release of GtH II. Although ovarian development was slightly stimulated by a combined T and GnRHa treatment, vitellogenesis was generally not initiated. The present study demonstrated that the juvenile striped bass pituitary is responsive to hormonal stimulation, resulting in elevated levels of GtH II in the pituitary and plasma. However, increased plasma levels of GtH II did not result in precocious puberty, suggesting that additional factors are required for the initiation of ovarian development in this teleost.     

Effects of steroids on GTH I and GTH II secretion and pituitary concentration in the immature rainbow trout Oncorhynchus mykiss

Using specific radio-immunoassays for rainbow trout GTH I and GTH II, the effects of testosterone and estradiol 17 beta have been studied or reinvestigated on the regulation of the secretion and the synthesis of the these two pituitary gonadotropins in the immature rainbow trout. After steroid implantation, the GTH II pituitary concentration is stimulated by testosterone and estradiol 17 beta for the entire period during which the plasma levels of these hormones are maintained to values comparable to those measured in the adult vitellogenic female rainbow trout. On the other hand, only testosterone induced a transient increase in the GTH I pituitary content 15 days after implantation, and estradiol provoked a decrease at day 30. The secretion of both GTH I and GTH II is stimulated by testosterone but not by estradiol 17 beta. Altogether, these results show that in the immature rainbow trout, testosterone preferentially modifies GTH I secretion, but not that of GTH II. They confirm that the stimulation of GTH II accumulation after testosterone or estradiol treatment would correspond to a stimulation of hormone synthesis. They evidence a differential action of both steroids on the synthesis of the two gonadotropins, especially a possible inhibition of GTH I synthesis by estradiol. They let suppose that the regulation of GTH I synthesis would involve factors other than steroids.     

Annual changes in serum vitellogenin concentrations in viviparous eelpout, Zoarces elongatus

Vitellogenin of matrotrophic viviparous eelpout (Zoarces elongatus) was purified from estradiol-17 beta (E2) treated immature male sera by gel chromatography and anion exchange chromatography. Isolated vitellogenin has a molecular weight of 540 kDa estimated by gel chromatography. Serum levels of vitellogenin in females were measured during oocyte development and gestation by single radial immunodiffusion. Serum vitellogenin level was low (less than 0.2 mg/ml) during the early vitellogenic period, increased in the late vitellogenic period to a peak level (6.4 +/- 2.1 mg/ml) at the beginning of gestation. After that it rapidly decreased to a low level (0.1 +/- 0.1 mg/ml) during the early gestation period. Levels of vitellogenin remained low throughout the gestation period. Serum E2 levels in females showed increased from 1.3 to 3.0 ng/ml during the late vitellogenic period, and declined to 0.4 ng/ml during the early gestation period. Serum levels of E2 showed good correlation with serum vitellogenin levels, suggesting that the vitellogenin synthesis is controlled by E2 in this species. These results combined with the matrotrophic growth of embryo during gestation suggest that there is a shift in the synthesis of maternal nutritional products for embryos from the yolk to other nutrients.     

Immunohistochemical detection of vitellogenin in male brown trout from Swiss rivers

The detection of vitellogenin, a yolk precursor protein, may serve as a biomarker for exposure to environmental oestrogens as its induction by xenobiotic oestrogens in the immature and male fish has been reported repeatedly. In the present work, juvenile brown trout were injected with oestradiol (5 microg g(-1) body weight oestradiol benzoate) in order to assess the induction and organ distribution of vitellogenin by means of immunohistochemistry. In addition, brown trout collected from Swiss rivers were analysed. Vitellogenin was detected in the oestradiol-injected juvenile trout but not in uninjected controls. The presence of vitellogenin was also demonstrated in a male and an immature feral brown trout from one of two locations downstream of three sewage treatment plants. In contrast, no positive staining was found in livers of trout upstream of the respective plants. The results demonstrate the suitability of immunohistochemistry for monitoring feral fish for the presence of vitellogenin production.     

Change in the management of per-subtrochanteric femoral fractures--a retrospective 10 year analysis

Renin-angiotensin system is present in mammalian ovarian follicular fluid and follicles. The demonstration of specific binding sites for angiotensin II in the follicular structures of rats suggests that angiotensin II may be related to the regulation of steroid biosynthesis in the mammalian ovary. Little is known about the presence and the action of angiotensin II in nonmammalian ovaries. An immunocytochemical investigation of angiotensin II in developing ovarian follicles of trout has been carried out. The specific antiserum was raised against (Val5)-angiotensin II of the trout. There was strong immunoreactivity in the ooplasm of endogenous vitellogenic follicles. Angiotensin II immunoreactive material was present in the follicular cells, but not within oocytes at the end of vitellogenesis. Immunocytochemically angiotensin II-like material in the ovary of rainbow trout fluctuates during the cycle of development.     

cDNA cloning of the beta subunit of teleost thyrotropin

cDNA clones encoding the beta subunit of thyrotropin (thyroid-stimulating hormone; TSH) were isolated from a cDNA library made from the pituitaries of immature rainbow trout and sequenced. The precursor of rainbow trout TSH beta consists of 147 aa, which can be cleaved into a signal peptide (20 aa) and a mature protein (127 aa) containing one potential N-glycosylation site and 12 cysteine residues. The protein showed highest homology with human TSH beta (51%) and lesser homology with human follitropin (42%), human lutropin (32%), and salmon gonadotropin (31-33%) beta subunits. The identification of TSH in addition to two gonadotropins (gonadotropins I and II) in the teleost fish suggests that the divergence of three kinds of glycoprotein hormones from an ancestral molecule took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods. Northern blot analysis showed that the expression of the rainbow trout TSH beta gene is specific to the pituitary gland and is significantly higher in immature fish than in mature fish, suggesting that TSH plays some role in the biological processes of immature fish.     

Gonadal in vitro androstenedione metabolism and changes in some plasma and gonadal steroid hormones during sex inversion of the protandrous sea bass, Lates calcarifer

Steroidogenesis in the gonad of the protandrous sea bass, Lates calcarifer, was examined in vitro in spermiating testis, previtellogenic ovary, and transitional gonads. Gonadal tissues were incubated with tritiated androstenedione. Metabolites were analyzed by thin-layer chromatography, high-performance liquid chromatography, microchemical reactions, and crystallization to constant specific activity. 17 beta-Hydroxysteroid dehydrogenase, 5 beta-reductase, and 3 alpha-hydroxysteroid dehydrogenase activities were found in all of the sex types. On the other hand, 11 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxylase activities were found only when testicular tissue was present, i.e., in testis and early transitional gonad. A low aromatase activity leading to estrone synthesis was detected in the previtellogenic ovary. In late transitional gonads, a major metabolite (metabolite X) was suggestively identified as a 3-ester of 17 beta-estradiol according to its chemical and immunological characteristics. Levels of 17 beta-estradiol (E2), the metabolite X, testosterone (T), and 11-ketotestosterone (11KT) were also measured by radioimmunoassay in plasma, before (January and February) and during (March and April) the sex inversion process. Plasma E2 was virtually undetectable (means below 25 pg/ml), although higher levels of metabolite X were found in transitional fish (485 +/- 432 pg/ml in March). Throughout this period, plasma levels of T and 11KT and the androgens/estrogens ratio were significantly higher in males than in transitional fish, where these levels decreased during the sex inversion period. The level of in vitro synthesis of metabolite X was high in transitional gonads, but their concentrations were very low (0.07 +/- 0.09 ng of equivalent E2/g in transitional gonads against 0.22 +/- 0.37 ng of equivalent E2/g in testes and 2.16 +/- 2.7 ng of equivalent E2/g in ovaries).     

Bisphenol A interacts with the estrogen receptor alpha in a distinct manner from estradiol

We investigated the interaction of bisphenol A (BPA, an estrogenic environmental contaminant used in the manufacture of plastics) with the estrogen receptor alpha (ERalpha) transfected into the human HepG2 hepatoma cell line and expanded the study in vivo to examine the effect of BPA on the immature rat uterus. Bisphenol A was 26-fold less potent in activating ER-WT and was a partial agonist with the ERalpha compared to E2. The use of ERalpha mutants in which the AF1 or AF2 regions were inactivated has permitted the classification of ER ligands into mechanistically distinct groups. The pattern of activity of BPA with the ERalpha mutants differed from the activity observed with weak estrogens (estrone and estriol), partial ERalpha agonists (raloxifene or 4-OH-tamoxifen), or a pure antagonist (ICI 182, 780). Intact immature female Sprague-Dawley rats were exposed to BPA alone or with E2 for 3 days. Unlike E2, BPA had no effect on uterine weight; however, like E2, both peroxidase activity and PR levels were elevated, though not to the level induced by E2. Following simultaneous administration, BPA antagonized the E2 stimulatory effects on both peroxidase activity and PR levels but did not inhibit E2-induced increases of uterine weight. These results demonstrate that BPA is not merely a weak estrogen mimic but exhibits a distinct mechanism of action at the ERalpha.     

Dopamine infusion does not alter LH levels before or after chronic cocaine exposure in female rhesus monkeys

Cocaine stimulates release of luteinizing hormone (LH) in preclinical and clinical studies but the contribution of the indirect dopamine agonist actions of cocaine to its effects on LH are unclear. In the present study, we examined the effects of exogenous dopamine infusions on LH release in drug-naive, normally cycling, female rhesus monkeys. All studies were conducted during the mid-follicular phase (cycle days 6-8). Three successive 80-min dopamine infusions (10 micrograms/kg/min, intravenous) were alternated with 20- or 40-min interruptions of dopamine infusions. There were no significant changes in LH during or following dopamine infusions. Predopamine baseline LH levels averaged 30 +/- 5.4 ng/ml. LH averaged 31.7 +/- 1.3 ng/ml during dopamine infusions and 31.4 +/- 1.3 ng/ml after dopamine infusions stopped. To determine whether chronic cocaine exposure influenced the effect of dopamine on LH, rhesus females were studied after more than 2 years of cocaine self-administration at an average dose of 6.5 +/- 0.2 mg/kg/day. LH averaged 27.3 +/- 3.3 ng/ml during baseline and 26.9 +/- 0.7 ng/ml and 26.1 +/- 0.7 ng/ml during dopamine infusions and interruptions, respectively. Similarly, during withdrawal from cocaine, baseline LH levels averaged 32.1 +/- 4.5 ng/ml, and LH did not change significantly during dopamine infusions (31.2 +/- 1.1 ng/ml) and infusion interruptions (32.1 +/- 1.1 ng/ml). Under the conditions of the present study, dopamine administration did not change LH levels in gonadally intact rhesus monkeys, and these findings are consistent with previous studies in ovariectomized rhesus females. However, these data are not consistent with clinical reports, and some possible implications of this species difference are discussed. Moreover, these data suggest that the stimulation of LH by cocaine may not be explained by its indirect dopamine agonist actions.     

The problem of asbestos today

In the past 2 years, a 4 year-old boy has had an anaphylactic reaction whenever he contacted food prepared with fish. The symptoms included intense itching in the throat and eyes, which progressed to generalized urticaria and facial angioedema. This was accompanied by cough, wheezing and dyspnea. Many fish preparations caused these episodes including several different kinds of fish (cod, tuna, salmon, trout, eel...), fish soup, chopsticks contaminated with fish preparations and canned fish. Elevated levels of total serum IgE (224 IU/ml) and specific IgE for cod (93.1 IU/ml), tuna (> 100 IU/ml), salmon (> 100 IU/ml), trout (64.4 IU/ml), mackerel (41.2 IU/ml) and eel (28.1 IU/ml) were found by the Pharmacia CAP system RAST FEIA in our allergy clinic. A skin prick test for mixed fish extracts (contain flounder, cod and halibut) was positive. A fish challenge test for cod, tuna, salmon, trout and eel all showed anaphylactic reactions. His allergic symptoms stabilized gradually after strictly avoiding ingestion of fish and using drug treatment. He also had a similar anaphylactic reaction to frogs. The best treatment for fish allergy is avoidance. Avoidance of fish may need to include both ingestion and inhalation of cooking vapors.     

Regulation of progesterone receptor messenger ribonucleic acid in the rat medial preoptic nucleus by estrogenic and antiestrogenic compounds: an in situ hybridization study

Progesterone receptor (PR) messenger RNA (mRNA) is concentrated in neurons of the preoptic area and other regions of the rat hypothalamus where it is colocalized with the estrogen receptor and regulated by changes in the steroid hormonal milieu. To date, little is known about the regulation of PR mRNA by estrogens and whether antiestrogenic compounds are capable of modulating its expression. The present studies used in situ hybridization to ascertain the time course of PR mRNA regulation in the medial preoptic nucleus by 17beta-estradiol, determine the effective dose required to elicit a response, and compare the efficacy of 17beta-estradiol with a variety of estrogenic or antiestrogenic compounds. The first series of studies revealed that the treatment of ovariectomized rats with 17beta-estradiol resulted in an increase in PR expression within 2 h, after which it remained elevated until 10 h postinjection and then returned to baseline levels. When ovariectomized rats were injected with 25-1000 ng/kg of 17beta-estradiol and euthanized 6 h later, a dose-dependent increase in the level of PR mRNA was observed, with a maximal response at 1000 ng/kg and an EC50 of 93.5 ng/kg. Subsequent studies evaluated the efficacy of a variety of estrogenic and antiestrogenic compounds in the rat preoptic nucleus. 17Beta-estradiol, diethylstilbestrol, and 17alpha-estradiol all significantly increased the level of PR mRNA, although the degree of induction varied with each compound. The injection of tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, GW 5638, or ICI 182,780 had no significant estrogenic effect on PR gene expression at the dose evaluated. In contrast, when tamoxifen or raloxifene, but not ICI 182,780, was administered in the antagonist mode, a significant dose-related decrease in the estradiol-induced level of PR mRNA was seen in the preoptic area. The results of these studies clearly demonstrate that PR mRNA expression in the rat preoptic area is rapidly stimulated by a small dose of 17beta-estradiol. Moreover, the present report has also shown that the estrogenic nature of compounds such as tamoxifen, raloxifene, toremifene, droloxifene, clomiphene, and GW 5638 cannot be predicted by their activity in peripheral tissues. Together, the results of these studies provide important information about the central activity of estrogens and provide evidence for their tissue-specifc actions in the rat.     

Inhibition of mitochondrial respiratory chain complex I by TNF results in cytochrome c release, membrane permeability transition, and apoptosis

Mitochondria have been shown to play a key role in apoptosis induction. However, the sequence of changes that occur in the mitochondria in the initial step of apoptosis has not been clearly elucidated. Here, we showed that mitochondrial respiratory chain (MRC) complex I was inhibited during the early phase of TNF- or serum withdrawal apoptosis. The importance of complex I inhibition in apoptosis is also supported by the observation that rotenone, an inhibitor of complex I but not that of other complexes, could induce apoptosis in a manner comparable to TNF. We hypothesized that inhibition of complex I could affect electron flow through other complexes leading to cytochrome c release by an antioxidant-sensitive pathway and caspase 3 activation followed by the induction of membrane permeability transition (MPT). This hypothesis is supported by the following observations: (1) TNF and rotenone induced MPT and cytochrome c release; (2) TNF-induced complex I inhibition was observed prior to cytochrome c release and MPT induction; (3) MPT induction was inhibited by a caspase 3 inhibitor, z-DEVD-CH2F, and an antioxidant pyrrolidine dithiocarbamate (PDTC), whereas cytochrome c release was only inhibited by PDTC. Thus, these results suggest that MRC complex I plays a key role in apoptosis signalings.     

Transcriptional activity of the hamster CYP11B2 promoter in NCI-H295 cells stimulated by angiotensin II, potassium, forskolin and bisindolylmaleimide

Interferon alpha (IFNalpha), a type I interferon, can be considered as a viral infection marker because this cytokine is induced during many viral infections. However, it is quite difficult to detect IFNalpha in sera. Investigations are interested in various intra-cellular IFNalpha-induced proteins as viral infection markers. However the activity of these enzymes increased not only in response to type I IFNs but also to type II IFN. MxA protein can be detected in the cytoplasm of IFNalpha/beta-treated cells, whereas other cytokines, including IFNgamma, are poor inducers. Using an immunochemiluminescent assay, we studied MxA protein in whole blood of 34 patients with various viral infections. The whole blood was drawn into sterile vacuum tubes containing heparin or EDTA. MxA values were relatively similar in heparin-treated samples and EDTA-treated samples, with differences not exceeding 1 ng/ml. The levels of MxA protein were compared in whole blood obtained by using two different lysis procedures. A correlation was found between the MxA levels obtained by using procedure I and procedure II, but higher amounts of MxA protein were found with procedure II. The second procedure is rapid and more convenient than the other and it is carried out in one step which reduce technical problems. High levels of MxA protein were found in peripheral blood cells of patients with acute viral infections (Rotavirus, Adenovirus, RSV, CMV), but MxA protein was not elevated in bacterial infections. The MxA levels were also studied in peripheral blood of 32 HCV positive patients. MxA protein was not found in most of IFNalpha-untreated patients, even those with high viral load. In contrast, high levels of MxA protein were found in IFNalpha-treated patients. MxA quantitation can be considered as a specific marker of acute viral infections, and could be useful in the management of treatment with IFNalpha.     

The circulating adhesion molecules sICAM-1 and sP-selectin in patients with sepsis

AIM: The aim of this study was to investigate whether the plasma levels of the circulating adhesion molecules sICAM-1 and sE-selectin could serve as early predictors of developing sepsis and its severity. METHODS: Twenty-four patients admitted to an intensive care unit with a high risk of developing septic complications were enrolled in this study. Patients were divided into three groups: group I, with infection without systemic sepsis, n = 8; group II, surviving patients with severe sepsis and multi-organ failure (MOF), n = 8; and group III, nonsurviving patients with severe sepsis and MOF, n = 8. Classification of patients was performed according to the clinical criteria defined by the Sepsis Consensus Conference in 1992. Blood samples were taken at 7 a.m. starting from the day of admission until the 7th day after diagnosis of sepsis. Plasma levels of sICAM-1 and sE-selectin were determined in all samples taken between the 3rd pre-septic day and the 7th day after the diagnosis of sepsis was made. RESULTS: In group I, both sICAM-1 (354.21 +/- 128.60 ng/ml, 86 samples) and sE-selectin (30.41 +/- 7.20 ng/ml, 86 samples) levels remained within the reference range over the whole period of observation. The sICAM-1 levels of group II (between 550.82 +/- 275.67 ng/ml and 445.08 +/- 243.63 ng/ml) tended to show values above the reference range without being significant. Mean sICAM-1 levels in group II did not differ from those of group I. From the 2nd pre-septic day onwards the sICAM-1 levels of group III increased, but not significantly. Significant differences in sICAM-1 levels between group I and group III were observed, with peaks at the samples of the 2nd preseptic day and after the 3rd day of sepsis, respectively (P < 0.05). The sE-selectin levels in group II were elevated from the 3rd preseptic day onwards, with a peak value on the 2nd day of sepsis (P < 0.05). Afterwards, levels decreased to initial values despite ongoing sepsis. Mean values of sE-selectin levels of group I and II were significantly different with the onset of sepsis (P < 0.05). Plasma levels of sE-selectin in group III were significantly elevated (66.30 +/- 9.00 ng/ml on the 3rd pre-septic day), reaching their maximal values of 106.67 +/- 21.66 ng/ml at the end of the observation period. Significant differences between sE-selectin levels of groups I and III existed from the 3rd pre-septic day onwards, and between group II and III on the 7th and 8th day of sepsis. CONCLUSION: Our results show that sICAM-1 is a relatively non-specific indicator for sepsis. In contrast, sE-selectin seems to be a good and early predictor of the beginning of severe sepsis with MOF. Furthermore, sE-selectin levels seem to have a prognostic value for the severity, possible course, and outcome of developing sepsis.     

Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland

The effect of hyperosmolality (300, 320 mosmol/kg H2O) and angiotensin II (A II, 10(-8) and 10(-6) M) was tested on a total of 64 neurons within the periventricular part of the anteroventral third ventricle (AV3V) region in brain slice preparations obtained from ovariectomized (OVX) rats with or without chronic treatment of estradiol-17 beta (E2). Hypertonic perfusion with a 320 mosmol/kg H2O but not a 300 mosmol/kg H2O medium caused a significant increase in the firing discharge rate in OVX animals. Perfusion with either hypertonic medium had no effect in E2-treated rats. The neuronal firing discharge rate of neurons in OVX rats was increased following perfusion with 10(-6) M A II, but not affected following perfusion with 10(-8) M A II. In E2-treated rats, perfusion with neither 10(-8) nor 10(-6) M A II had any effect. These data suggest a dynamic relationship between the ovarian endocrine function and the central mechanisms regulating dipsogenic behavior and/or release of vasopressin in the female rat.     

Adrenomedullin, amylin, calcitonin gene-related peptide and their fragments are potent inhibitors of gastric acid secretion in rats

A release of radio-immunoassayable LHRH from the stalk-median eminence of neonatal piglets and prepubertal gilts was measured using an in vitro incubation system. The stalk-median eminence was collected from one-week-old male (n = 19) and female (n = 21) piglets and from 6-month-old prepubertal ovariectomized gilts given oestradiol benzoate (20 micrograms/kg b.w.; n = 52) or left untreated (control; n = 25) 30 or 68 h before slaughter. Each vial, containing the stalk-median eminence in 2 ml of Krebs-Ringer bicarbonate buffer, was incubated for 30 min, followed by 30 min incubations during which either basal release or the effect of adrenoreceptor antagonists and agonists on LHRH output was evaluated. There were no differences between the basal release of LHRH (x +/- SEM; pg/ml) from the stalk-median eminence of male (65.5 +/- 9.8) and female (66.3 +/- 9.6) newborn piglets. The addition of propranolol (10(-6) M) caused a 250% increase in LHRH release from the stalk-median eminence explants of neonatal males (p = 0.08) and females (p < 0.05). Neither norepinephrine nor phentolamine affected LHRH release from the stalk-median eminence of newborn males and females. The basal release of LHRH (pg/ml) from the stalk-median eminence explants collected from ovariectomized gilts given oestradiol benzoate 30 and 68 h before slaughter or left untreated was similar (147.5 +/- 36.1, 236.4 +/- 77.7 and 202.0 +/- 41.6, respectively). Propranolol evoked a significant increase in LHRH secretion from the stalk-median eminence in the control group, but not in the groups given oestradiol benzoate. Norepinephrine (10(-6) M) increased LHRH release from the stalk-median eminence collected from the control animals, 30 h and 68 h after oestradiol benzoate treatment by 48, 78 and 73 percent, respectively. Phentolamine (10(-6) M) did not affect LHRH release from the stalk-median eminence in control animals and ovariectomized gilts primed with oestradiol benzoate. Urapidil (10(-6) M, alpha 1-adrenoreceptor antagonist) did not affect the basal LHRH release from the stalk-median eminence of gilts from the control group and group slaughtered 30 h after oestradiol benzoate treatment, but caused a rapid increase of LHRH release from the stalk-median eminence 68 h after oestradiol benzoate treatment. Phenylephrine (10(-6) M) did not affect LHRH output from the stalk-median eminence collected at various time periods after oestradiol benzoate administration in vivo. These results suggest that in pigs, nerve terminals releasing LHRH at the stalk-median eminence level are sensitive to adrenergic stimulation or inhibition and that the adrenergic system can be modulated by estrogens in the prepubertal gilts.     

Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle

Brains of the gilthead seabream, Sparus aurata, contain three different forms of gonadotropin-releasing hormone (GnRH): seabream (sb) GnRH, chicken (c) GnRH-II, and salmon (s) GnRH. In the present study, we developed three specific enzyme-linked-immunosorbent assays (ELISA) for sbGnRH, cGnRH-II, and sGnRH and used them to measure the levels of each GnRH form in the pituitary of male and female seabream at different stages of gametogenesis. The sensitivity was 6 pg/well for the sbGnRH assay, 7 pg/well for the cGnRH-II assay, and 2 pg/well for the sGnRH assay. Levels of each of the three GnRH forms were measured in pituitaries from fish sampled at the beginning of gonadal recrudescence and during the spawning season. Of the three forms, only sbGnRH and cGnRH-II were detected in the pituitary, irrespective of reproductive state or sex. Recrudescent fish had similar levels of sbGnRH and cGnRH-II in the pituitary. In sexually mature fish, the levels of sbGnRH were higher than those in recrudescent fish while pituitary cGnRH-II content remained unchanged. Consequently, sbGnRH levels were 3- to 17-fold higher than cGnRH-II levels in mature fish. Positive correlations also existed between pituitary sbGnRH content and pituitary and plasma gonadotropin (GtH) II levels. Surprisingly, mature 1-year-old males had significantly higher levels of sbGnRH in the pituitary than mature 3-year-old males, while pituitary and plasma GtH II levels were similar between these two groups. Although the reason for this difference in sbGnRH levels is unclear, a possible role of sbGnRH in the processes of puberty or sex-inversion is implied. Based on the present results, it can be suggested that in the gilthead seabream, sbGnRH is the most relevant form of GnRH in the control of reproduction.     

The deleterious effects of exogenous angiotensin I and angiotensin II on myocardial ischemia-reperfusion injury

Angiotensin II is well known to have a cardiotoxic effects. However, it is still unclear whether exogenous angiotensin I or angiotensin II has a deleterious effect on myocardial ischemia-reperfusion injury. To examine this deleterious effects, we administered angiotensin I and angiotensin II to perfused hearts before ischemia, and measured creatine kinase (CK) release and cardiac function during subsequent reperfusion. Wistar Kyoto rats were used and the hearts were perfused by the Langendorff technique at a constant flow (10 ml/min). Seven hearts were perfused for 20 min and then subjected to 15 min of global ischemia (Control). In the experimental groups, during the 5 min before ischemia, we administered 100 ng/ml angiotensin I (Ang I; n = 9), 1 microgram/ml enalaprilat (ACEI; n = 5), both agents (ACEI + Ang I) (n = 6), or 10 ng/ml angiotensin II (Ang II; n = 6). The perfusates were then sampled to measure angiotensin II. After 15 min of ischemia, the hearts were reperfused with control perfusate. Throughout the 20 min of reperfusion, the effluent was collected to measure cumulative CK release. Angiotensin I increased coronary perfusion pressure (CPP) by 32 +/- 4 mmHg, however, the angiotension converting enzyme inhibitor inhibited the increase of CPP by angiotension I (11 +/- 1 mmHg) (p < 0.01). The contents of angiotensin II in the effluent in Ang I and Ang I + ACEI were 11.5 +/- 1.9 ng/ml and 4.0 +/- 0.5 ng/ml (p < 0.01). After 20 min of reperfusion, the left ventricular developed pressure was unchanged in all of the groups. CPP was also unchanged by ischemia in all of the groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reproductive success of female rainbow trout (Oncorhynchus mykiss) in response to graded dietary ascorbyl monophosphate levels

Ascorbic acid is an essential nutrient in rainbow trout diets and has been shown to play an important role in fish reproduction. Recommended dietary levels are based on immature fish, and the specific requirements for brood stock are unknown. To establish the optimum dietary level for mature rainbow trout, six graded levels of ascorbyl-2-monophosphate were fed to groups of female fish over a period of 10 mo until spawning. Increasing dietary levels of ascorbyl monophosphate resulted in significantly increased ascorbic acid concentrations in liver, kidney, ovaries, and ovulated eggs. Liver and egg concentrations were saturable at 109.3 and 266.6 micrograms ascorbic acid/g tissue, respectively. Tissue saturation levels of 83.7% and 91.2%, respectively, were reached at the highest dietary level (870 mg ascorbyl monophosphate/kg diet) tested. Both fecundity and embryo survival increased significantly with dietary ascorbyl monophosphate levels. The results indicated that the present National Research Council recommended dietary level of 50 mg ascorbic acid/kg diet for rainbow trout is inadequate for brood stock fish. An amount 8 times higher is necessary to optimize tissue ascorbic acid levels and achieve maximum reproductive success.     

Effects of captopril related to increased levels of prostacyclin and angiotensin-(1-7) in essential hypertension

OBJECTIVE: To evaluate the contribution of angiotensin-(1-7) [Ang-(1-7)] and prostaglandins to the acute and long-term antihypertensive actions of captopril in mild-to-moderate essential hypertensive patients. DESIGN AND METHODS: Blood pressure, cardiac rate and the plasma concentrations of angiotensin I (Ang I), angiotensin II (Ang II), Ang-(1-7), prostaglandin E2 and 6-keto prostaglandin F1 alpha (the breakdown product of prostacyclin) were determined in the peripheral venous blood of 24 essential hypertensive subjects before and 3 h after administration of 50 mg captopril. Eleven of 24 patients completed a 6-month treatment period with captopril monotherapy (50 mg twice a day). The hemodynamic and hormonal response produced by a last 50 mg dose of captopril was determined once again in the 11 subjects who maintained blood pressure control with captopril monotherapy for 6 months. RESULTS: The fall in blood pressure produced 3 h after drug intake was comparable for the first and the last 50 mg captopril dose. Although the first response to captopril increased plasma levels of Ang I only, the response to the last dose of the drug (6 months after) caused significantly higher levels of Ang I and Ang-(1-7). Neither acute nor chronic therapy with captopril had a significant effect on plasma concentrations of Ang II. Although plasma levels of prostaglandin E2 and 6-keto prostaglandin F1 alpha were not modified by a first exposure to captopril, the concentrations of 6-keto prostaglandin F1 alpha but not prostaglandin E2 rose significantly in subjects treated with the inhibitor for 6 months. A negative correlation was also demonstrated between diastolic blood pressure and plasma Ang-(1-7) levels in the 11 essential hypertensive subjects in whom blood pressure was controlled with captopril monotherapy. CONCLUSIONS: Inhibition of angiotensin converting enzyme with captopril had a significant effect on blood pressure that was not directly accounted for by a suppression of plasma Ang II levels. Continuous therapy with captopril unmasked a contribution of Ang-(1-7) and prostacyclin to the antihypertensive actions of this drug.