Molecular cloning and expression analysis of a novel caspase recruitment domain protein (CARD) in common carp Cyprinus carpio L

A novel caspase recruitment domain protein (CARD) was isolated from common carp Cyprinus carpio L. by expressed sequence tag analysis. This gene consist of a 2016 bp open reading frame and untranslated regions, which is putatively translated to a protein of 535 amino acid residues. The gene harbors domains (CARD and Coiled-coil domain), which are conserved in proteins of CARD family. The CARD domain have carp was similar to human CARD9 with 72.4% identity. Expression analysis revealed that CARD gene of carp (carp-CARD) expressed in normal tissues of head kidney, spleen, liver, heart and brain. Here we demonstrated that the expression of carp-CARD increased by cortisol treatment in all the tissues and had a high and long lasting expression in cortisol treated spleen.     

Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.)

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33 days post hatch (dph), while the spleen was the last organ to be detected at 49 dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66 dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66 dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94 dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.     

Steroids,histamine, and serotonin in the medicinal leech salivary gland secretion

Lipids represent 20% of the total weight of the dried pool of medicinal leech salivary gland secretion (SGS) obtained from about 50 individual animals. SGS lacks phospholipids, but contains steroids. Immunochemiluminescent analysis of SGS revealed the presence of free steroid hormones: cortisol, progesterone, testosterone, estradiol, and dehydroepiandrosterone. Micro-chromatographic-mass spectrometric analysis of SGS and its low molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standard preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione, and testosterone) and histamine and serotonine have been identified in SGS.     

Suppression in function of phagocytic cells in common carp Cyprinus carpio L. injected with estradiol,progesterone or 11-ketotestosterone

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone, progesterone and cortisol was analysed in common carp (Cyprinus carpio L.). Carp were injected intraperitoneally (1 and 5 microg/kg fish) with each steroid and the activity of kidney macrophages was recorded 1 and 3 days post-injection. Bovine serum albumin-injected fish served as controls. Phagocytosis, superoxide anion and nitric oxide production were suppressed in all sex steroid treatments, and the suppression was in a dose-dependent manner. Cortisol treatment also inhibited the immune parameters to an extent similar to each of the steroid hormone. These results show that sex steroids can modulate the function of the phagocytic cells.     

In vivo treatment with progestogens causes immunosuppression of carp Cyprinus carpio leucocytes by affecting nitric oxide production and arginase activity

In this study, carp Cyprinus carpio were injected with various steroid compounds, including synthetic and natural progestogens and the glucocorticoid cortisol, to investigate effects on leucocytes isolated from their kidneys. Injection of cortisol led to an increased spleeno-somatic index (I(S)) on day 21 post-injection (pi) and immunosuppressive effects measured as decreased nitric oxide (NO) production and increased arginase activity in isolated leucocytes on days 14 and 21 pi, respectively. Moreover, reduced NO production was also observed after injection of the synthetic progestogens, levonorgestrel (LEV) and medroxyprogesterone acetate. In addition, LEV influenced arginase activity in head kidney cells on day 14 and day 21 pi. This study is the first demonstration in fishes that the application of these steroid compounds in vivo affects NO production and arginase activity of isolated leucocytes.     

Gender differences in the vasomotor effects of different steroid hormones in rat pulmonary and coronary arteries.

It is well recognised that oestrogens possess vasodilatory properties, and similar responses to testosterone have been demonstrated. However, vasomotor effects of other steroid hormones have not been well described. Direct comparisons of the relative vasoactivity of different steroid hormones in different vascular beds in male and female genders have not been made. Coronary and pulmonary arteries from adult Wistar rats were mounted in a wire myograph, loaded to 100 and 17 mmHg respectively, maximally pre-contracted with 1 x 10(-4) M prostaglandin-F-2-alpha, and dose response curves to 1 x 10(-6) to 1 x 10(-3) or 3 x 10(-3) M of 17 beta-oestradiol, testosterone, progesterone, and cortisol dissolved in water were constructed. Addition of each steroid hormone caused acute, dose dependent dilatation in coronary and pulmonary vessels. In coronary arteries the order of activity was testosterone > progesterone > 17 beta-oestradiol > cortisol, p < 0.001. In pulmonary arteries, the order of activity was progesterone > testosterone > cortisol > 17 beta-oestradiol, p < 0.001. Pulmonary arteries from male animals were more sensitive to the effects of testosterone than those from female animals, p = 0.003, whereas coronary arteries from female animals were more sensitive to the effects of 17 beta-oestradiol than those from male animals, p < 0.001. We have demonstrated significant differences in the in vitro vasomotor effects of different steroid hormones in two distinct vascular beds. Gender differences in vasomotor responses to steroid hormones may play a role in the aetiology of vasospastic diseases.     

Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone and progesterone was analyzed in common carp Cyprinus carpio. Carp kidney leukocytes were cultured in RPMI 1640 medium containing 0.1, 1, 10, 100 or 1000 nM concentration of each hormone. The production of superoxide anion, nitric oxide (NO) and phagocytosis were measured in vitro. Similar concentrations of cortisol were used as control. Phagocytic activities of carp macrophages was suppressed by treatment with beta-estradiol, progesterone and 11-ketotestosterone. The production of NO in carp macrophages was suppressed by progesterone and 11-ketotestosterone. However, carp macrophages incubated with beta-estradiol, progesterone and 11-ketotestosterone did not show any difference in the production of superoxide anion in comparison with control macrophages in the absence of hormones. Carp macrophages treated with cortisol suppressed phagocytosis and the production of nitric oxide and superoxide anion.     

Effects of graded levels of dietary methionine hydroxy analogue on immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian)

Immune response and antioxidant status of immune organs in juvenile Jian carp (Cyprinus carpio var. Jian) fed graded levels of methionine hydroxy analogue (MHA) (0, 5.1, 7.6, 10.2, 12.7, 15.3 g kg(-1) diet) for 60 days were investigated. Results indicated that head kidney index, spleen index, red and white blood cell counts significantly increased with increasing MHA levels up to a point (P < 0.05), whereupon decreased (P < 0.05). Glutathione reductase activity in head kidney and spleen, anti-hydroxy radical and glutathione-S-transferase activities in spleen, catalase activity and GSH content in head kidney significantly increased by MHA supplement, while malondialdehyde content, anti-superoxide anion, superoxide dismutase, glutathione peroxidase activities in head kidney and spleen, protein carbonyl content and catalase activity in spleen, anti-hydroxy radical activity in head kidney significantly decreased by MHA supplement. However, protein carbonyl content and glutathione-S-transferase activity in head kidney, GSH content in spleen remained unaffected. After 60-day feeding trial, a challenge study was conducted by injection of Aeromonas hydrophila for 17 days. Results showed that survival rate, leukocytes phagocytic activity, lysozyme activity, acid phosphatase activity, total iron-binding capacity, haemagglutination titre, complement 3, 4 and immunoglobulin M contents significantly increased by optimal dietary MHA supplement (P < 0.05). These data suggested that MHA affected antioxidant status of immune organs and promoted immune response in juvenile Jian carp.     

Gender‐ and season‐dependent relationships between testosterone, oestradiol and immune functions in wild roach

Plasma testosterone and 17β‐oestradiol concentrations, differential leukocyte counts and proportion of dead Rhipidocotyle campanula gill parasites (parasite resistance) were determined five times during a year in two populations of roach Rutilus rutilus and analysed for seasonal and gender differences. In addition to the above immune variables, plasma Immunoglobulin M (IgM) concentration, chemiluminescence and migration differential of head kidney phagocytes, size of the spleen, haematocrit and total leukocyte count were correlated with sex hormones for each population, sampling time and sex separately, using condition factor as a partial correlate. There were no clear gender differences in the determined immune variables. Both testosterone and oestradiol concentrations were lowest after the spawning in June. Oestradiol was higher in females than in males, but testosterone was present in equal concentrations in both sexes. Statistically significant correlations between sex hormones and immune variables were rare and mainly without any patterns with respect to population, sex or sampling date. The meta‐analysis on individual correlations, however, revealed a positive relationship of plasma testosterone concentration to chemiluminescence of head kidney phagocytes, plasma IgM concentration and the proportion of dead R. campanula to all R. campanula on the gills. In the meta‐analysis, the concentration of oestradiol was not found to correlate with any of the studied immune variables.     

ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.     

Studies on lipoprotein and adrenal steroidogenesis: II. Utilization of low density lipoprotein- and high density lipoprotein-cholesterol for steroid production in functioning human adrenocortical adenoma cells in culture

We examined the utilization of human low density lipoprotein (LDL)- and high density lipoprotein (HDL)-cholesterol for steroid production in primary monolayer culture cells from adenomas of primary aldosteronism and Cushing's syndrome and an adrenal of nodular hyperplasia of Cushing's syndrome. We compared the data obtained with findings in the case of cultured normal human adrenocortical cells. In the presence of 10(-7) M adrenocorticotropin (ACTH), the addition of either LDL or HDL to the culture medium at a cholesterol concentration of 100 micrograms/ml led to a significant increase in the daily secretion rates of cortisol, dehydroepiandrosterone sulfate (DHEA-S) and aldosterone in the adenoma and nodular hyperplasia cells, as in the normal cells. Although LDL greatly increased the secretion of steroid hormones, no significant difference in steroid secretion following the treatments with LDL and HDL were observed in these cultured cells. The contribution of endogenous cholesterol to steroid production was also high, thereby indicating that the neoplastic transformation did not have untoward effects. Cells from adenomas of primary aldosteronism secreted not only aldosterone, but also cortisol and DHEA-S. The daily secretion rates of these steroids were markedly increased when ACTH was added to the medium. With prolonged exposure to ACTH, however, the rate of aldosterone secretion showed a gradual decrease with the incubation time. This decrease might be due to the impaired conversion of corticosterone to 18-hydroxycorticosterone. In case of adenomas in patients with Cushing's syndrome, the secretion of steroid hormones varied in quantity and quality, depending on the type of plasma cortisol response to the rapid ACTH test in vivo, thereby suggesting that the adrenocortical adenoma of Cushing's syndrome might be divided into two subtypes. These results indicate that human functioning adrenocortical adenoma cells utilize plasma lipoproteins as a source of cholesterol for steroidogenesis during the prolonged stimulation of steroid secretion.     

Cortisol affects testicular development in male common carp, Cyprinus carpio L., but not via an effect on LH secretion

Previous work showed that prolonged elevated cortisol levels, implicated in the stress adaptation, inhibits testicular pubertal development in male common carp, as well as an impairment of the synthesis of the 11-oxygenated androgens. This may be a direct effect of cortisol on the testis or via the gonadotropin secretion by the pituitary. The aim of the present study was to investigate whether cortisol has an effect on pituitary LH secretion. Juvenile common carp were fed with cortisol containing food pellets. Elevated cortisol levels blocked the increase in testosterone levels and pituitary LH content, but induced higher plasma LH levels at the end of puberty. The in vitro LH release capacity was correlated to the pituitary LH content. At the final stage of pubertal development, when a significant difference in pituitary LH content was observed, sGnRHa-induced LH release was also decreased. Testosterone has been shown to induce development of pituitary gonadotrophs, leading to an increase in LH content and GnRH-inducible LH release, but a decrease in plasma LH levels. We observed decreased plasma testosterone levels as a consequence of prolonged cortisol treatment. It is hypothesised that cortisol inhibits the testicular testosterone secretion and thereby, prevents LH storage. In vitro, this leads to a reduced GnRH-inducible LH release, but in vivo to increased LH plasma levels. It is very unlikely that the impaired testicular development is due to an effect of cortisol on LH secretion.     

Corticosteroids affect the testicular androgen production in male common carp (Cyprinus carpio L.).

Our previous experiments to study the effect of stress adaptation on pubertal development in carp showed that repeated temperature stress and prolonged feeding with cortisol-containing food pellets, which mimics the endocrine stress effects, retarded the first waves of spermatogenesis and decreased 11-ketotestosterone (11KT) plasma levels. The objective of the present study was to investigate whether the decrease in plasma 11KT is caused by a direct effect of cortisol on the steroid-producing capacity of the testis or by an indirect effect, such as a decrease in plasma LH. Pubertal and adolescent isogenic male common carp (Cyprinus carpio L.) were fed with either cortisol-containing food pellets or control food pellets over a prolonged period. Our results indicate that cortisol has a direct inhibitory effect on the testicular androgen secretion independent of the LH secretion. Furthermore, the pubertal period is critical to the influence of cortisol regarding testicular androgen secretion, because the effect is no longer observed at adolescence.     

草鱼肾脏和脾脏血细胞发育过程的观察

鱼类肾脏、脾脏是机体造血的主要器官。印迹涂片显示:草鱼肾脏和脾脏内血细胞的发育过程大致经历了三个阶段,即原始阶段、幼稚阶段、成熟阶段。本文着重描述了各阶段细胞的形态特征井对草鱼血细胞的发育及命名等问题作了初步的探讨。       

Specificity of the effect of growth hormone on DNA concentration in the nuclei of the lymphoid cells of hypophysectomized rats]

The effect of growth hormone on the DNA content in the nuclei of the thymus, spleen and the lymph node lymphocytes was studied by means of cytophotometry. In hypophysectomized rats the growth hormone increased the DNA content in the nuclei of the middle lymphocytes of these organs without altering its amount in the small lymphocytes. Thymus lymphocytes were the most sensitive to the hormone action. The DNA content in the nuclei of these cells increased as soon as one hour after the administration of the hormone; in 4 hours it reached the maximum. Other hormones with an anabolic effect (insulin, thyroxin, testosterone), induced no elevation of DNA in the thymocyte nuclei at that period of time. A conclusion was drawn on the high tropicity of the growth hormone to the cells of the lymphoid organs and particularly to the thymocytes (middle lymphocytes of the thymus).     

Ramadan fasting alters endocrine and neuroendocrine circadian patterns. Meal-time as a synchronizer in humans?

Muslims must refrain from eating, drinking, smoking, and sexual relations from sunrise to sunset during the month of Ramadan. Serum concentrations of melatonin, steroid hormones (cortisol, testosterone), pituitary hormones (prolactin, LH, FSH, GH, TSH) and thyroid hormones (free thyroxin and free triiodothyronine) were documented around the clock at six 4-hourly intervals before Ramadan began and on the twenty-third day of Ramadan (daytime fasting). Time series were analysed with repeated measures ANOVA. Statistically significant differences were found in some variables: the nocturnal peak of melatonin was diminished and may have been delayed; there was a shift in the onset of cortisol and testosterone secretion; the evening peak of prolactin was enhanced, FSH and GH rhythmic patterns were affected little or not at all by Ramadan fasting and only the serum TSH rhythm was blunted over the test time span. These data show that daytime fasting, modifications in sleep schedule and psychological and social habits during Ramadan induce changes in the rhythmic pattern of a number of hormonal variables.