Acute daily exposure to ambient air pollution impairs endothelium-dependent vasodilator responses in young, healthy individuals.

Exposure to ambient air pollution influences cardiovascular (CV) morbidity and mortality. The differential effects of changing particulate or gaseous air pollution on endothelial function in young healthy individuals remain unclear. The aim of this study was to evaluate the relationships between exposures to different pollutants and vascular function in a group of 39 young (33±11 years old) subjects with low CV risk. Flow-mediated dilatation (FMD) and nitroglycerin-mediated dilatation (NMD) were performed, when air pollution reached highest levels (heating period) and repeated in a subgroup of 18 participants a few months later (just before the heating period starts). Daily mean concentrations of PM2.5 and PM10 were inversely correlated with FMD, and this relationship remained significant after adjusting for factors known to affect vascular dysfunction. Endothelial function did not differ between the two time points studied. However, we observed a strong inverse association between the change in the concentration of particulate matter (deltaPM2.5 and deltaPM10) and the change in FMD (deltaFMD) between the two visits (R= -0.65, p= 0.02; R= -0.64, p= 0.02, respectively). In summary, we provide evidence that the concentration of PM2.5 and PM10, but not SO2, NO, NO2, CO, or O3 is associated with impaired endothelial function in young, healthy individuals.

Systemic and vascular inflammation in experimental allergic asthma.

Allergic asthma and atherosclerosis are inflammatory diseases characterized by similar sets of circulating inflammatory cells, in addition to mast cells in the airway and vessel wall. Animal models and human studies provide evidence of a potential interaction between the two apparently unrelated diseases. The main objective of this study was to determine whether experimental allergic asthma is accompanied by inflammatory responses, measured as the activation of the vasculature and the presence of immune cells in the perivascular adipose tissue. For this purpose, male Dunkin Hartley guinea pigs weighing 250 - 300 g were sensitized twice with 10 µg ovalbumin dissolved in aluminium hydroxide (Al(OH)3). Allergen inhalation was performed 10 days after the second immunization and continued 5 days a week for 2 months. After that period, T cell and macrophage content was measured by flow cytometry. The aortic expression of inflammatory markers was studied by real-time PCR. The number of T cells in the peripheral blood was significantly greater in the allergic group in comparison to the sham group. We did not find any significant differences in the leukocyte content of the perivascular adipose tissue between the groups. Nor did we identify significant changes in the expression of inflammatory markers (tumor necrosis factor, monocyte chemoattractant protein-1) and adhesion molecules (intercellular adhesion molecules and vascular cell adhesion molecules) in the aorta. Interestingly, we observed a significantly decreased expression of the endothelial nitric oxide synthase (eNOS) mRNA in the aortic vessel of the allergic group compared to the sham group.

Nox1/4 inhibition exacerbates age dependent perivascular inflammation and fibrosis in a model of spontaneous hypertension.

Hypertension is associated with oxidative stress and perivascular inflammation, critical contributors to perivascular fibrosis and accelerated vascular ageing. Oxidative stress can promote vascular inflammation, creating options for potential use of NADPH oxidase inhibitors in pharmacological targeting of perivascular inflammation and its consequences. Accordingly, we characterized age-related changes in oxidative stress and immune cell infiltration in normotensive (WKY) and spontaneously hypertensive rats (SHRs). Subsequently, we used pharmacological inhibitors of Nox1 (ML171) and Nox1/Nox4 (GKT137831; 60 mg/kg), to modulate NADPH oxidase activity at the early stage of spontaneous hypertension and investigated their effects on perivascular inflammation and fibrosis. RESULTS: Ageing was associated with a progressive increase of blood pressure as well as an elevation of the total number of leukocytes, macrophages and NK cells infiltrating perivascular adipose tissue (PVAT) in SHRs but not in WKY. At 1 month of age, when blood pressure was not yet different, only perivascular NK cells were significantly higher in SHR. Spontaneous hypertension was also accompanied by the higher perivascular T cell accumulation, although this increase was age independent. Aortic Nox1 and Nox2 mRNA expression increased with age only in SHR but not in WKY, while age-related increase of Nox4 mRNA in the vessels has been observed in both groups, it was more pronounced in SHRs. At early stage of hypertension (3-months) the most pronounced differences were observed in Nox1 and Nox4. Surprisingly, GKT137831, dual inhibitor of Nox1/4, therapy increased both blood pressure and perivascular macrophage infiltration. Mechanistically, this was linked to increased expression of proinflammatory chemokines expression (CCL2 and CCL5) in PVAT. This inflammatory response translated to increased perivascular fibrosis. This effect was likely Nox4 dependent as the Nox1 inhibitor ML171 did not affect the development of spontaneous hypertension, perivascular macrophage accumulation, chemokine expression nor adventitial collagen deposition. In summary, spontaneous hypertension promotes ageing-associated perivascular inflammation which is exacerbated by Nox4 but not Nox1 pharmacological inhibition.

Oxidative stress and inflammatory markers in prediabetes and diabetes.

Prediabetes is a state of elevated plasma glucose in which the threshold for diabetes has not yet been reached and can predispose to the development of type 2 diabetes and cardiovascular diseases. Insulin resistance and impaired beta-cell function are often already present in prediabetes. Hyperglycemia can upregulate markers of chronic inflammation and contribute to increased reactive oxygen species (ROS) generation, which ultimately cause vascular dysfunction. Conversely, increased oxidative stress and inflammation can lead to insulin resistance and impaired insulin secretion. Proper treatment of hyperglycemia and inhibition of ROS overproduction is crucial for delaying onset of diabetes and for prevention of cardiovascular complications. Thus, it is imperative to determine the mechanisms involved in the progression from prediabetes to diabetes including a clarification of how old and new medications affect oxidative and immune mechanisms of diabetes. In this review, we discuss the relationship between oxidative stress and hyperglycemia along with links between inflammation and prediabetes. Additionally, the effects of hyperglycemic memory, microvesicles, micro-RNA, and epigenetic regulation on inflammation, oxidative state, and glycemic control are highlighted. Adipose tissue and their influence on chronic inflammation are also briefly reviewed. Finally, the role of immune-targeted therapies and anti-diabetic medication on glycemic control and oxidative stress are discussed.

Effects of controlled physical activity on immune cell phenotype in peripheral blood in prehypertension - studies in preclinical model and randomised crossover study.

Hypertension (HT) is a global public health issue. There are many behavioural risk factors including unhealthy diet, tobacco use and alcohol consumption as well physical inactivity that contribute to the development of high blood pressure (BP) and its complications. Favourable effect of regular physical activity on treatment or prevention of hypertension by improvement of endothelial function is widely accepted however little is known about its relationship with immune system. Thus, the aim of this study was to assess the role of moderate regular physical activity on immune cell phenotype. T cell and monocyte subsets were characterised in 31 subjects with prehypertension (130 - 139 mmHg systolic and 85 - 89 mmHg diastolic blood pressure) who participated in moderate training (3 times/week) on cyclometers for 3 months in crossover study design. Complementary study was performed in murine model of Ang II-induced hypertension and ten-week-old animals were trained on a treadmill (5 times/week, 1 hour) for 2 weeks before and 1.5 weeks after minipumps implantation. In the context of elevated blood pressure regular physical activity had modest influence on immune cell phenotype. Both in human study and murine model we did not observe effects of applied exercise that can explain the mechanism of BP reduction after short-term regular training. Twelve-weeks regular training did not affect the activation status of T lymphocytes measured as expression of CD69, CD25 and CCR5 in human study. Physical activity resulted in higher expression of adhesion molecule CD11c on CD16+ monocytes (especially CD14 high) without any changes in leukocytes subpopulation counts. Similar results were observed in murine model of hypertension after the training. However the training caused significant decrease of CCR5 and CD25 expressions (measured as a mean fluorescence intensity) on CD8+ T cells infiltrating perivascular adipose tissue. Our studies show modest regulatory influence of moderate training on inflammatory markers in prehypertensive subjects and murine model of Ang II induced hypertension.

Controlled aerobic training improves endothelial function and modifies vascular remodeling in healthy adults with high normal blood pressure.

The aim of the study was to assess endothelial function in adults with high normal blood pressure (HNBP) undergoing controlled aerobic training. The study was conducted among 31 volunteers with HNBP. Subjects underwent supervised cycle ergometer training for 12 weeks. Exercise intensity was assessed by monitoring the pulse with intention to keep the heart rate increase within the range of 40% to 65% of the heart rate reserve. The control group consisted of 14 healthy adults, not subjected to any intervention. The control group was examined twice at 12-week intervals (non-exercising time control). Vascular endothelial function was determined by flow-mediated dilation (FMD) and by measuring total nitric oxide products (NOx). The measurement of carotid intima-media complex thickness (IMT) was an indirect method of assessing vascular remodeling. Blood pressure (ABPM method), anthropological parameters and lipid profile were also assessed. There was a significant change in FMD after 3-month training in the study group: the average FMD training was 5.21 ± 2.17%, while after the program FMD increased to 9.46 ± 3.69% (P < 0.001). After training, the NOx also increased from 1.01 ± 0.38 µmol/L to 1.27 ± 0.48 µmol/L (P < 0.001). Effects were observed irrespective of participants' sex. Interestingly, a modest but significant reduction of IMT was also observed, from 0.5 ± 0.06 mm to 0.46 ± 0.10 mm (P = 0.04). There was also a reduction in the percentage of body fat content from 25.01 ± 8.77% to 22.31 ± 8.79% (P < 0.001). No statistically significant changes were noted after 12 weeks of training in the blood pressure and lipid profile. In the control group no statistically significant changes of any parameter were observed. Regular aerobic exercise improves nitric oxide-dependent endothelial function of the vessels and can initiate regression of atherosclerosis in people with HNBP.

Anti-atherosclerotic effect of the angiotensin 1-7 mimetic AVE0991 is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis.

BACKGROUND AND PURPOSE: Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1-7 (Ang-(1-7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1-7) mimetics, such as AVE0991. The mechanisms underlying the vaso-protective effects of AVE0991, a Mas receptor agonist, remain to be explored. EXPERIMENTAL APPROACH: We investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)-/- mice, in the context of vascular inflammation and plaque stability. KEY RESULTS: AVE0991 has significant anti-atherosclerotic properties in ApoE-/- mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoE-/- mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1ß, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. CONCLUSIONS AND IMPLICATIONS: The selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoE-/- mice. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

Heterogeneity of peripheral blood monocytes, endothelial dysfunction and subclinical atherosclerosis in patients with systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by increased cardiovascular morbidity and mortality. SLE patients have increased prevalence of subclinical atherosclerosis, although the mechanisms of this observation remain unclear. Considering the emerging role of monocytes in atherosclerosis, we aimed to investigate the relationship between subclinical atherosclerosis, endothelial dysfunction and the phenotype of peripheral blood monocytes in SLE patients. METHODS: We characterized the phenotype of monocyte subsets defined by the expression of CD14 and CD16 in 42 patients with SLE and 42 non-SLE controls. Using ultrasonography, intima-media thickness (IMT) of carotid arteries and brachial artery flow-mediated dilation (FMD) as well as nitroglycerin-induced dilation (NMD) were assessed. RESULTS: Patients with SLE had significantly, but only modestly, increased IMT when compared with non-SLE controls (median (25th/75th percentile) 0.65 (0.60/0.71) mm vs 0.60 (0.56/0.68) mm; p < 0.05). Importantly, in spite of early atherosclerotic complications in the studied SLE group, marked endothelial dysfunction was observed. CD14dimCD16+proinflammatory cell subpopulation was positively correlated with IMT in SLE patients. This phenomenon was not observed in control individuals. Interestingly, endothelial dysfunction assessed by FMD was not correlated with any of the studied monocyte subsets. CONCLUSIONS: Our observations suggest that CD14dimCD16+monocytes are associated with subclinical atherosclerosis in SLE, although the mechanism appears to be independent of endothelial dysfunction.

Characterization of the impairment of the uptake of apoptotic polymorphonuclear cells by monocyte subpopulations in systemic lupus erythematosus.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16- cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

Vasodilatory effect and endothelial integrity in papaverine- and milrinone-treated human radial arteries.

Prevention of the vasospasm is an important aspect of coronary artery bypass grafting (CABG) with the use of radial artery (RA) as the conduit. We compared the effect of two phosphodiesterase inhibitors papaverine and milrinone on vasodilation and endothelial integrity of human RA segments harvested from 20 CABG patients. Vasodilatory effect of the drugs were assessed by organ bath technique in RA rings precontracted with KCl and phenylephrine. Endothelial integrity was evaluated by CD34 immunofluorescence in frozen sections. Vasorelaxation induced by papaverine was significantly greater as compared to that induced by milrinone (90.47% ± 10.16% vs. 78.98% ± 19.56%, p<0.05). Similarly, pretreament with papaverine more strongly inhibited the contractile response of RA rings to KCl (6.0 ± 8.0 mN vs. 26.7 ± 21.5 mN, p<0.001). Papaverine was also superior to milrinone in the preservation of endothelial integrity (75.3% ± 12.9% vs. 51.8% ± 18.0%, p<0.02). In conclusion, papaverine seems to be more suitable than milrinone for prevention of vasospasm in radial artery conduits used for CABG.

Disruption of the secretion and action of 17,20ß-dihydroxy-4-pregnen-3-one in response to a rise in temperature in the Arctic charr, Salvelinus alpinus. Consequences on oocyte maturation and ovulation.

Plasma levels of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP), and the timing of ovulation were investigated in female Arctic charr (Salvelinus alpinus) reared at 5°C and at 10°C during the pre-spawning period. The effects of switching from 5 to 10°C, and from 10 to 5°C were also investigated. 17,20ßP plasma levels were higher at 5°C than at 10°C. A switch from 10 to 5°C stimulated 17,20ßP secretion, whereas a switch from 5 to 10°C had the opposite effect. Ovulation occurred spontaneously in the females kept at 5°C, and in those switched from 10 to 5°C. In contrast, ovulation was inhibited in females reared at 10°C, and in those switched from 5 to 10°C. Oocyte maturation at 5°C and at 10°C in the presence of LH or of 17,20ßP was also investigated in vitro using donor females reared at 5 or 10°C. Both LH and 17,20ßP stimulated oocyte maturation more effectively in oocytes incubated at 5°C than at 10°C. At both incubation temperatures, the rearing temperature of the donor females had a significant impact on their responsiveness to LH stimulation, but had no effect on their responsiveness to 17,20ßP stimulation. In addition to the inhibition of LH secretion, which had already been reported, the results reported here show that in Arctic charr raising the temperature above the physiological range reduces both follicular responsiveness to LH stimulation and the sensitivity of oocytes to 17,20ßP stimulation.

Effect of UVA and 8-methoxypsoralen, 4, 6, 4'-trimethylangelicin or chlorpromazine on apoptosis of lymphocytes and their recognition by monocytes.

Photopheresis (ECP) is an immunomodulatory therapy that involves extracorporeal exposure of isolated peripheral blood leukocytes to UVA irradiation in the presence of 8-methoxypsoralen (8-MOP) followed by their reinfusion to the patient. However, the underlying mechanism of ECP is not well understood yet. We selected 8-methoxypsoralen (8-MOP), chlorpromazine (CPZ) and 4,6,4'-trimethylangelicine (TMA) because of differences in their ability to induce immune suppression in rats in vivo. In this study, we investigated the role of UVA irradiation of lymphocytes in the presence of TMA, CPZ or 8-MOP on cell apoptosis, and their impact on adhesion of lymphocytes to monocytes in vitro. Apoptosis of lymphocytes and their sub-populations (lymphocytes T and B, NK cells) were determined by a flow cytometry, using AnnexinV-FITC, TUNEL assay and DNA content analysis and antibodies CD3, CD56, CD19. Mitochondrial potential was measured using CMXRos staining and the interaction of monocytes with lymphocytes was monitored by PKH26 Red Sigma staining of lymphocytes and subsequent use of flow cytometry. Our results show a significant increase of apoptosis of the photochemically treated lymphocytes and a decrease of their mitochondrial potential that depended on the dose and time after the treatment. Our data also reveal an increased recognition of apoptotic lymphocytes by freshly isolated monocytes.

Influence of long-term exposure to dietary cadmium on growth, maturation and reproduction of goldfish (subspecies: Prussian carp Carassius auratus gibelio B.).

The influence of long-term exposure of goldfish to dietary cadmium (Cd) on its accumulation in tissues, growth, ovarian development, luteinizing hormone (LH) secretion and a response to hormonal stimulation of spawning were evaluated. The study was conducted on four groups of females for the period of 3 years, from the age of 10 weeks to second spawning. Four doses of Cd were applied in the feed: 0 (control group), 0.1, 1 and 10 mg Cd g(-1) of feed (wet weight). The highest dose of Cd (10 mg g(-1)) inhibited growth and caused several behavioural effects. In contrast, lower dose of Cd (1 mg g(-1)) stimulated fish growth. The doses of Cd from 0.1 to 1 mg Cd g(-1) did not influence ovarian development. The gonado-somatic index (GSI) and histological analysis of ovaries showed no differences in ovarian development between the control group and the groups receiving these doses of Cd. However, in the group receiving the highest Cd dose, GSI decreased. This was associated with persistent, long-lasting elevation of plasma LH levels. Ovulation did not occur in this group. Injections of salmon GnRH-analogue (sGnRHa) alone or with domperidone (a dopamine receptor antagonist) in sexually mature fish caused an increase of LH levels in all groups, although in the group fed with the highest Cd dose the effect was weaker than in the other groups. After the first spawning season, a negative effect of lower Cd doses (0.1 and 1mg Cd g(-1)) on ovarian recrudescence (rebuilding of ovaries) and on the response to the consecutive hormonal stimulation of spawning was observed (lower number of ovulating females). There was a significantly higher content of Cd in the livers of fish than in their muscles. The results of hormonal stimulation of spawning and histological analysis of ovaries suggest that in goldfish cadmium acts mainly at the level of ovary rather than on the pituitary gland. We suppose that in the natural environment cadmium present in the feed can play an important role in the accumulation of this element in fish tissues and can influence vital physiological processes.

Temperature effects along the reproductive axis during spawning induction of grass carp (Ctenopharyngodon idella).

For grass carp (Ctenopharyngodon idella) raised in the Ivory Coast (with water temperatures of 26-31 degrees C), induced spawning is obligatory for fry production. However, ovulation rates following hormonal treatment are often low. We hypothesized that high temperatures are an inhibiting factor for the reproductive axis (brain-pituitary-gonad) in these conditions. By in vivo and in vitro experiments, we tried to determine the thermosensitive steps during spawning induction. We compared gonadotropin and maturation-inducing steroid (MIS) profiles during a spawning induction at controlled temperatures of 24 and 28 degrees C in relation to ovulation success. We performed pituitary cell cultures and ovarian fragment incubations at controlled temperatures. The ovulation rate was lower at 28 degrees C (10%) than at 24 degrees C (36%). At the pituitary level, we found only minor thermal impacts on GnRH-stimulated LH release, but our data suggest an increase of the dopaminergic inhibition by high temperatures. The main effects were found at the ovary level, where ovary responsiveness to gonadotropin by MIS synthesis was disturbed, as well as oocyte responsiveness to MIS triggering final maturation, and probably ovulation. These results show the importance of regulating temperature during spawning induction to ensure a high rate of ovulation.

The influence of gabaergic drugs upon LH secretion from the pituitary of the common carp: an in vitro study.

In the present study, the perifusions of whole pituitary glands of spermiating male common carp were performed in the presence of several GABAergic drugs. Muscimol (agonist of GABA(A) receptors) and bicuculline (the antagonist of the same type of GABA receptors) did not modify basal LH release. LH basal secretion was not modified when pituitaries were perifused with baclofen--an agonist of GABAB receptors. On the other hand, baclofen at doses of 10(-8) and 10(-4) M significantly decreased GnRH-A-induced LH release to about 86% and 88% of LH levels in control group, respectively. In our previous study we have shown that GABA decreased basal and GnRH-A-stimulated in vivo and in vitro LH release. In conclusion, it can be suggested that in the mature male carp GABA exerts an inhibitory influence on GnRH-stimulated LH release, probably through the inhibition of the GnRH action on gonadotropes. This inhibition seems to be mediated by the B type of GABA receptors.

Post-ovulatory secretion of pituitary gonadotropins GtH I and GtH II in the rainbow trout (Oncorhynchus mykiss): regulation by steroids and possible role of non-steroidal gonadal factors.

In order to determine the factors of ovarian origin which can modulate the postovulatory secretion of the FSH-like gonadotropin (GtH I) and the LH-like gonadotropin (GtH II), freshly ovulated female rainbow trout were divided into two groups. In the first group the fish were stripped in order to eliminate the eggs and ovarian fluid from the body cavity, while in the second group the eggs were kept in the body cavity. Subsequently, fish from both groups were implanted with testosterone (10 mg/kg), 17beta-estradiol (10 mg/kg) or 17,20beta-ddihydroxy-4-regnen-3-one (17,20betaP) (1 mg/kg) or injected every 2 days with desteroidized ovarian fluid (1.5 ml/kg). The secretion of GtH I dramatically increased in stripped fish, reaching its maximum levels 2 weeks after ovulation. The preservation of eggs in the body cavity led to the suppression of this increase. The profiles of GtH II secretion were opposite to those encountered for GtH I because the increase of GtH II was observed only in unstripped fish. The administration of steroids showed that testosterone is able to inhibit GtH I release and stimulate that of GtH II in stripped fish, having no effect on the release of these gonadotropins in non-stripped animals. 17beta-Estradiol failed to modify GtH I secretion, however it decreased the release of GtH II in fish containing retained eggs in the body cavity. 17,20betaP had a delayed stimulating influence on GtH I release in unstripped fish. Finally, multiple injections of desteroidized ovarian fluid into stripped fish led to a significant decrease of GtH I release and to an increase of GtH II secretion. This study demonstrates that factors, which are present in ovarian fluid, modulate the post-ovulatory secretion of both gonadotropins--their net action is negative on GtH I and positive on GtH II. Among the steroids, testosterone is of major importance, being able to inhibit GtH I release and to stimulate that of GtH II. We also show that non-steroidal factors present in the ovarian fluid can influence the release of both gonadotropins, which indirectly supports the previous findings about the existence of inhibin/activin-like factors in fish.

GTH I and GTH II secretion profiles during the reproductive cycle in female rainbow trout: relationship with pituitary responsiveness to GnRH-A stimulation.

The recent purification of two gonadotropins, GTH I and GTH II, in teleost fish and the development of their specific radioimmunoassays using antibodies directed against their beta subunits have demonstrated that earlier assays for GTH II also measured GTH I. Most of the results on the gonadotropic control of reproduction in fish must thus be reinvestigated using specific assays for each gonadotropin. The present investigation examines changes in blood plasma levels of GTH I and GTH II during the annual reproductive cycle of rainbow trout in relation to the ability of gonadotropin-releasing hormone (GnRH) to stimulate in vivo GTH I and GTH II secretion, with focus on the periovulatory period. GTH I was detected from immature to postovulatory stages, with a significant increase at the onset of exogenous vitellogenesis, with GTH I levels rising from 7.83 +/- 3.37 to 16.87 +/- 4.52 ng/ml. GTH II remained very low until the end of the vitellogenesis. For both hormones, the most significant variations were measured during the periovulatory period. GTH II levels peaked on the day of maturation, but the increase was biphasic with a first peak arising 4 days prior to maturation. This evaluation of GTH II was preceded by a progressive and significant rise GTH I levels starting from 5.83 +/- 2.17 ng/ml 8 days before maturation and increasing to more than 10 ng/ml on the day of maturation. Thus, the GTH II maturation surge is not the only gonadotropic signal occurring before ovulation. The role of the preovulatory GTH I increase remains unknown. After ovulation the secretory profiles of the two hormones depended on the presence of absence of ovulated eggs in the body cavity. There was a major increase in GTH I levels starting 4 days after ovulation and egg stripping, reaching more than 25 ng/ml. Conversely, in these fish the GTH II levels gradually decreased. In the fish which kept their eggs in the body cavity the progress was reversed; 8 days after maturation, GTH II increased to levels similar to those measured prior to maturation; the presence of the eggs prevented an increase in GTH I. This seems to indicate that postovulatory regulation of GTH I and GTH II secretion might involve ovarian factors that act in an antagonistic fashion. The prevention of the increase in GTH I levels in the presence of eggs suggests that as long as eggs are present in the body cavity, the development of a new cycle of gametogenesis is not possible, since GTH I is the gonadotropin mainly involved in controlling this phenomena. GnRH cannot significantly stimulate GTH I secretion at any stage of gametogenesis, even when its levels increased after ovulation. Other factors antagonizing GnRH are involved. The well-known antagonistic effect of dopamine on the GnRH stimulated GTH II secretion is fish is not involved since the dopamine antagonist, pimozide, was ineffective in inducing a stimulatory action of GnRH on GTH I secretion. Although GnRH can stimulate GTH II secretion from mid-vitellogenesis, the response to GnRH was not correlated with GTH II in blood. These results suggest that GTH I and GTH II secretions are regulated by different mechanisms and different factors.

A radioimmunoassay for African catfish growth hormone: validation and effects of substances modulating the release of growth hormone.

A highly sensitive radioimmunoassay has been developed for measuring plasma growth hormone (GH) concentrations in the African catfish (Clarias gariepinus). The lower detection limit of the assay was 0.1 ng/ml and the standard curve had an ED50 value of 0.5 ng/ml. The validity of the assay was established and the effects of several neurotransmitters on the release of GH were examined. In vitro experiments, using a static culture system for dispersed pituitary cells, demonstrated that the GH release in African catfish was affected by growth hormone-releasing hormone and somatostatin. Single intraperitoneal injections with a dopamine agonist, apomorphine, produced significant and dose-dependent increases in plasma GH levels. Unlike carp, goldfish, and tilapia, a super-active analogue of salmon gonadotrophin-releasing hormone did not alter plasma GH levels in African catfish.

Effects of N,N-dimethylnitrosamine (DMNA) on in vitro oocyte maturation and embryonic development of fertilized eggs of carp (Cyprinus carpio L.) kept in eutrophied ponds.

Toxic effects of N,N-dimethylnitrosamine (DMNA) at doses of 100 or 500 micrograms l-1 on in vitro carp oocyte maturation (steroidogenesis), embryonic development and hatching of larvae (obtained as a result of artificial spawning of females kept for four seasons in normal and eutrophicated ponds) were investigated. There were no significant effects of DMNA on oocyte maturation and steroidogenesis during 24 h of incubation. The DMNA decreased the hatching of fertilized eggs derived from control females. This decrease reached a level of significance at a dose of 500 micrograms l-1. However, the effect of long-term exposure of female fish to eutrophied water was very much higher. The trend of the in vitro DMNA effect was the same, but it did not reach a statistically significant level. The results suggest that nitrosamines, through their effect on egg hatchability, may reduce fish populations along with increasing aquatic eutrophication.

Neuropeptide Y stimulates in vivo gonadotropin secretion in teleost fish.

The effects of the neuropeptide Y (NPY), alone or in combination with a gonadotropin-releasing hormone analogue, D-Ala6-desGly10-Pro9-Net LHRH (LHRHa), have been studied on the in vivo secretion of the maturational gonadotropin (GtH2) in the rainbow trout Oncorhynchus mykiss and the common carp Cyprinus carpio. Depending on the species, two routes of administration were used: in trout, intraperitoneal injection (20 micrograms/kg body wt); in carp, direct infusion (3 micrograms/kg body wt) into the third ventricle via a temporary brain cannula. In both cases NPY alone induced a twofold increase in GtH2 secretion and peaked 2 to 4 hr administration regardless of the route of injection. The plasma gonadotropin levels returned to basal within 8 hr. The relative increases (peaked secretion/basal secretion) did not differ with the route of injection. When the animals were first treated with NPY and then LHRHa (20 micrograms/kg) 1 hr later, the magnitude of the response to LHRHa was greater in the animals pretreated with NPY, indicating either a potentiation of LHRHa action by NPY or additive effects of the two peptides. The return to basal levels also took longer in fish receiving NPY first. NPY may act directly at the pituitary level or activate central neuromediatory systems.