11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Marine sponges of the order Verongida are a rich source of biologically active bromotyrosine-derived secondary metabolites. However, none of these compounds are known to display anti-inflammatory activity. In the present investigation, we report the anti-inflammatory effects of 11-oxoaerothionin isolated from the Verongida sponge Aplysina fistularis. When RAW264.7 cells and primary macrophages were preincubated with 11-oxoaerothionin and stimulated with LPS (lipopolysaccharide), a concentration-dependent inhibition of iNOS (inducible nitric oxide synthase) protein and NO2- (Nitrite) production were observed. The same effect was observed when proinflammatory cytokines and PGE(2) (Prostaglandin E2) production was evaluated. In summary, we demonstrated that in the presence of LPS, 11-oxoaerothionin suppresses NO2 and iNOS expression as well as inflammatory cytokines and PGE(2).

Temporal relationships of a pulse of prolactin (PRL) to a pulse of a metabolite of PGF2 alpha in mares

Hourly blood samples were collected from 10 mares during 24 h of each of the preluteolytic, luteolytic, and postluteolytic periods. The autocorrelation function of the R program was used to detect pulse rhythmicity, and the intra-assay CV was used to locate and characterize pulses of prolactin (PRL) and a metabolite of prostaglandin F2 alpha (PGFM). Rhythmicity of PRL and PGFM concentrations was detected in 67% and 89% of mares, respectively. Combined for the three periods (no difference among periods), the PRL pulses were 5.2 +/- 0.4 h (mean +/- SEM) at the base, 7.5 +/- 1.5 h between nadirs of adjacent pulses, and 12.3 +/- 1.5 h from peak to peak. The peaks of PRL pulses were greater (P < 0.05) during the luteolytic period (46 +/- 14 ng/mL) and postluteolytic period (52 15 ng/mL) than during the preluteolytic period (17 3 ng/mL). Concentrations of PRL during hours of a PGFM pulse were different (P < 0.003) within the luteolytic period and postluteolytic period and were greatest at the PGFM peak; PRL concentrations during a PGFM pulse were not different during the preluteolytic period. The frequency of the peak of PRL and PGFM pulses occurring at the same hour (synchrony) was greater for the luteolytic period (65%, P < 0.01) and postluteolytic period (50%, P < 0.001) than for the preluteolytic period (17%). This is the first report in mares on characterization and rhythmicity of PRL pulses, synchrony between PRL and PGFM pulses, and greater PRL activity during the luteolytic and postluteolytic periods than during the preluteolytic period. (C) 2012 Elsevier Inc. All rights reserved.

Ovarian and PGF2 alpha responses to stimulation of endogenous PRL pulses during the estrous cycle in mares

The effects of a PRL-stimulating substance (sulpiride) on PRL and PGF2 alpha secretion and on luteal and ovarian follicular dynamics were studied during the estrous cycle in mares. A control group (n = 9) and a sulpiride group (Sp; n = 10) were used. Sulpiride (25 mg) was given every 8 h from Day 13 postovulation to the next ovulation. Repeated sulpiride treatment did not appear to maintain PRL concentrations at 12-h intervals beyond Day 14. Therefore, the hypothesis that a long-term increase in PRL altered luteal and follicular end points was not testable. Hourly samples were collected from the hour of a treatment (Hour 0) to Hour 8 on Day 14. Concentrations of PRL increased to maximum at Hour 4 in the Sp group. The PRL pulses were more prominent (P < 0.008) in the sulpiride group (peak, 19.4 +/- 1.9 ng/mL; mean +/- SEM) than in the controls (11.5 +/- 1.8 ng/mL). Concentrations of a metabolite of PGF2a (PGFM), number, and characteristics of PGFM pulses, and concentrations of progesterone during Hours 0 to 8 were not affected by the increased PRL. A novel observation was that the peak of a PRL pulse occurred at the same hour or 1 h later than the peak of a PGFM pulse in 8 of 8 PGFM pulses in the controls and in 6 of 10 pulses in the Sp group (P < 0.04), indicating that sulpiride interfered with the synchrony between PGFM and PRL pulses. The hypothesis that sulpiride treatment during the equine estrous cycle increases concentrations of PRL and the prominence of PRL pulses was supported. (c) 2012 Elsevier Inc. All rights reserved.

Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

Background: Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Methods: Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results: GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. Conclusions: The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Abstract Background Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Variations in maternal care alter corticosterone and 17beta-estradiol levels, estrous cycle and folliculogenesis and stimulate the expression of estrogen receptors alpha and beta in the ovaries of UCh rats

Background: Variations in maternal care are associated with neonatal stress, hormonal disturbances and reproductive injuries during adulthood. However, the effects of these variations on sex hormones and steroid receptors during ovary development remain undetermined. This study aimed to investigate whether variations in maternal care are able to influence the hormonal profile, follicular dynamics and expression of AR, ER-alpha and ER-beta in the ovaries of UCh rat offspring. Methods: Twenty-four adult UCh rats, aged 120 days, were randomly divided into two groups (UChA and UChB) and mated. Maternal care was assessed from birth (day 0) to the 10th postnatal day (PND). In adulthood, twenty adult female rats (UChA and UChB offspring; n = 10/group), aged 120 days, were euthanized by decapitation during the morning estrus. Results: UChA females (providing high maternal care) more frequently displayed the behaviors of carrying pups, as well as licking/grooming and arched back nursing cares. Also, mothers providing high care had elevated corticosterone levels. Additionally, offspring receiving low maternal care showed the highest estrous cycle duration, increased corticosterone and 17beta-estradiol levels, overexpression of receptors ER-alpha and ER-beta, increased numbers of primordial, antral and mature follicles and accentuated granulosa cell proliferation. Conclusions: Our study suggests that low maternal care alters corticosterone and 17beta-estradiol levels, disrupting the estrous cycle and folliculogenesis and differentially regulating the expression of ER-alpha and ER-beta in the ovaries of adult rats.

Morfologia das células intersticiais de ovários policísticos de ratas: um estudo experimental

OBJETIVOS: Avaliar a histomorfometria das células intersticiais dos ovários, bem como analisar a concentração sanguínea de esteroides sexuais de ratas portadoras de ovários policísticos induzidos pela luz contínua. MÉTODOS: Vinte ratas foram divididas em dois grupos: ratas na fase de estro (GCtrl ) e ratas portadoras de ovários policísticos induzidos pela iluminação contínua (GOP). Os animais do GCtrl permaneceram com período de luz das 7:00 s 19:00 horas, e os animais do GOP, com iluminação contínua (400 Lux), durante um período de 60 dias. Ao final desse período todos os animais foram anestesiados, foi coletado o sangue, para determinação dos níveis séricos de estradiol (E2), progesterona (P4) e testosterona (T), seguido da retirada dos ovários que foram fixados em formol a 10% e processados para inclusão em parafina. Cortes histológicos com 5 µm corados pela hematoxilina e eosina foram utilizados para análise histomorfométrica. As análises morfológicas, contagem de cistos, determinação da concentração e do volume nuclear das células intersticiais foram realizadas com o auxílio de microscópio de luz adaptado a uma câmera de alta resolução (AxioCam), cujas imagens foram transmitidas e analisadas em computador com software AxioVision Rel 4.8 (Carl Zeiss). Os dados obtidos foram submetidos ao teste t de Student (p<0,05). RESULTADOS: A morfologia mostrou a presença de cistos nos ovários pertencentes ao Grupo OP e de corpos lúteos no GCtrl, mostrando ainda evidências da origem das células intersticiais a partir das células da teca interna desses cistos. Com relação aos níveis hormonais o GOP apresentou níveis séricos de estradiol (pg/mL) aumentados em relação ao GCtrl (GOP=124,9±4,2>GCtrl=73,2±6,5; p<0,05), o mesmo ocorrendo com os níveis de testosterona (pg/mL) (GOP=116,9±4,6>GCtrl=80,6±3,9; p<0,05). Entretanto os níveis de progesterona (ng/mL) foram mais elevados no GCtrl em relação ao GOP (GCtrl=16,3±2,0>GOP=4,2±1,5; p<0,05). A morfometria mostrou haver aumento significante do volume nuclear no grupo GOP (GOP=102,1±5,2>GCtrl=63,6±16,5; p<0,05), assim como da área ocupada (%) pelas células intersticiais (GOP=24,4±6,9>GCtrl=6,9±3,2; p<0,05) em relação aos animais do GCtrl. CONCLUSÃO: As células intersticiais do ovário policístico da rata provavelmente provêm dos cistos ovarianos devido degeneração das células da granulosa e diferenciação das células da teca interna. As elevações dos níveis séricos de testosterona e de estradiol provavelmente provêm do aumento significativo da atividade celular e da área ocupada pelas células intersticiais.

Morfologia dos cardiomiócitos e quantificação do colágeno no miocárdio de ratas tratadas com isoflavonas ou estrogênios

OBJETIVOS: Avaliar a morfologia dos cardiomiócitos e quantificar o colágeno presente no miocárdio de ratas tratadas com extrato concentrado de soja ou 17β-estradiol (E2). MÉTODOS: Vinte e oito ratas foram divididas em quatro grupos: GCtrl - fase de estro; GOvx - ovariectomizadas (Ovx); GIso - Ovx tratadas com extrato de soja (150 mg/kg, por dia); GE2 - Ovx tratadas com E2 (10 µg/kg, por dia). As drogas e o veículo (0,2 mL de propilenoglicol) foram administrados após 30 dias da realização da ovariectomia, por 30 dias consecutivos. No último dia os animais foram anestesiados, o coração retirado, mergulhado em formaldeído a 10%, e fragmentos dos ventrículos submetidos a processamento histológico, sendo os cortes corados pela hematoxilina e eosina ou pelo picrosirius-red. As análises histomorfométricas (contagem, volume nuclear e quantificação do colágeno) foram realizadas em microscópio de luz e software AxioVision Rel. 4.2, sendo o colágeno determinado pelo programa Imagelab 2000. Os dados foram submetidos ao teste de ANOVA complementado pelo teste de Tukey (p<0,05). RESULTADOS: Notamos maior quantidade de núcleos de cardiomiócitos nos animais dos grupos Ovx e Iso do que no GE2 e GCtrl (GOvx=121,7±20,2=GIso=92,8±15,4>GE2=70,5±14,8=GCtrl=66,3±9,6; p<0,05), sendo o volume nuclear maior nos animais do grupo Ctrl e E2 (GE2=35,7±4,8=GCtrl=29,9±3,6>GIso=26,5±4,5=GOvx=22,4±2,9; p<0,05). Com relação ao colágeno notamos maior concentração no grupo Ovx (GOvx=5,4±0,1>GCtrl=4,0±0,1=GIso=4,4±0,08=GE2=4,3±0,5; p<0,05). CONCLUSÕES: Os estrogênios previnem a diminuição do volume nuclear dos cardiomiócitos e a deposição de colágeno entre as fibras musculares cardíacas. Já a administração de isoflavonas previne somente a deposição de colágeno, o que pode preservar as propriedades mecânicas das fibras cardíacas.

Applying many-body expansion aiming to make ab in initio molecular dynamics more efficient

In this present work we present a methodology that aims to apply the many-body expansion to decrease the computational cost of ab initio molecular dynamics, keeping acceptable accuracy on the results. We implemented this methodology in a program which we called ManBo. In the many-body expansion approach, we partitioned the total energy E of the system in contributions of one body, two bodies, three bodies, etc., until the contribution of the Nth body [1-3]: E = E1 + E2 + E3 + …EN. The E1 term is the sum of the internal energy of the molecules; the term E2 is the energy due to interaction between all pairs of molecules; E3 is the energy due to interaction between all trios of molecules; and so on. In Manbo we chose to truncate the expansion in the contribution of two or three bodies, both for the calculation of the energy and for the calculation of the atomic forces. In order to partially include the many-body interactions neglected when we truncate the expansion, we can include an electrostatic embedding in the electronic structure calculations, instead of considering the monomers, pairs and trios as isolated molecules in space. In simulations we made we chose to simulate water molecules, and use the Gaussian 09 as external program to calculate the atomic forces and energy of the system, as well as reference program for analyzing the accuracy of the results obtained with the ManBo. The results show that the use of the many-body expansion seems to be an interesting approach for reducing the still prohibitive computational cost of ab initio molecular dynamics. The errors introduced on atomic forces in applying such methodology are very small. The inclusion of an embedding electrostatic seems to be a good solution for improving the results with only a small increase in simulation time. As we increase the level of calculation, the simulation time of ManBo tends to largely decrease in relation to a conventional BOMD simulation of Gaussian, due to better scalability of the methodology presented. References [1] E. E. Dahlke and D. G. Truhlar; J. Chem. Theory Comput., 3, 46 (2007). [2] E. E. Dahlke and D. G. Truhlar; J. Chem. Theory Comput., 4, 1 (2008). [3] R. Rivelino, P. Chaudhuri and S. Canuto; J. Chem. Phys., 118, 10593 (2003).

Estradiol induces transcriptional and posttranscriptional modifications in versican expression in the mouse uterus

We have previously shown the differential expression of versican in the mouse uterus under ovarian hormone influence. We also demonstrated there is not a direct correlation between mRNA levels and protein expression, suggesting posttranscriptional events, such as alteration in mRNA stability. This posttranscriptional effect may result in the elongation and stabilization of transcripts poly(A) tail. Thus, the aim of this study was to analyze whether estradiol (E2) regulates versican mRNA stability and expression in a dose-related and time-dependent manner. For this purpose female mice were ovariectomized and treated with a single injection of 0.1 or 10 μg E2. To block transcription a group of females received a single injection of alpha-amanitin before hormone administration. Uterine tissues were collected 30 min, 1, 3, 6, 12 and 24 h after treatments and processed for quantitative real time PCR (qPCR), RACE-PAT Assay and immunohistochemistry. qPCR showed that versican mRNA levels are higher than control from 3 to 24 h after E2 administration, whereas after transcription inhibition versican mRNA unexpectedly increases within 3 h, which can be explained when transcriptional blockers alter the degradation rate of the transcript, resulting in the superinduction of this mRNA. Accordingly, analysis of versican transcript poly(A) tail evidenced a longer product 3 h after treatment, but not after 12 h. Versican immunoreaction becomes conspicuous in the superficial stroma only 3 h after E2 injection, whereas the whole stroma is immunoreactive from 6 h onward. These results demonstrate that E2 modulates versican at the transcriptional and posttranscriptional levels in a time-dependent manner.

Formaldehyde inhalation reduces respiratory mechanics in a rat model with allergic lung inflammation by altering the nitric oxide/cyclooxygenase-derived products relationship

Bronchial hyperresponsiveness is a hallmark of asthma and many factors modulate bronchoconstriction episodes. A potential correlation of formaldehyde (FA) inhalation and asthma has been observed; however, the exact role of FA remains controversial. We investigated the effects of FA inhalation on Ovalbumin (OVA) sensitisation using a parameter of respiratory mechanics. The involvement of nitric oxide (NO) and cyclooxygenase-derived products were also evaluated. The rats were submitted, or not, to FA inhalation (1%, 90 min/day, 3 days) and were OVA-sensitised and challenged 14 days later. Our data showed that previous FA exposure in allergic rats reduced bronchial responsiveness, respiratory resistance (Rrs) and elastance (Ers) to methacholine. FA exposure in allergic rats also increased the iNOS gene expression and reduced COX-1. L-NAME treatment exacerbated the bronchial hyporesponsiveness and did not modify the Ers and Rrs, while Indomethacin partially reversed all of the parameters studied. The L-NAME and Indomethacin treatments reduced leukotriene B4 levels while they increased thromboxane B2 and prostaglandin E2. In conclusion, FA exposure prior to OVA sensitisation reduces the respiratory mechanics and the interaction of NO and PGE2 may be representing a compensatory mechanism in order to protect the lung from bronchoconstriction effects.

Humoral autoimmune response heterogeneity in the spectrum of primary biliary cirrhosis

Objective To compare autoantibody features in patients with primary biliary cirrhosis (PBC) and individuals presenting antimitochondria antibodies (AMAs) but no clinical or biochemical evidence of disease. Methods A total of 212 AMA-positive serum samples were classified into four groups: PBC (definite PBC, n = 93); PBC/autoimmune disease (AID; PBC plus other AID, n = 37); biochemically normal (BN) individuals (n = 61); and BN/AID (BN plus other AID, n = 21). Samples were tested by indirect immunofluorescence (IIF) on rat kidney (IIF-AMA) and ELISA [antibodies to pyruvate dehydrogenase E2-complex (PDC-E2), gp-210, Sp-100, and CENP-A/B]. AMA isotype was determined by IIF-AMA. Affinity of anti-PDC-E2 IgG was determined by 8 M urea-modified ELISA. Results High-titer IIF-AMA was more frequent in PBC and PBC/AID (57 and 70 %) than in BN and BN/AID samples (23 and 19 %) (p < 0.001). Triple isotype IIF-AMA (IgA/IgM/IgG) was more frequent in PBC and PBC/AID samples (35 and 43 %) than in BN sample (18 %; p = 0.008; p = 0.013, respectively). Anti-PDC-E2 levels were higher in PBC (mean 3.82; 95 % CI 3.36–4.29) and PBC/AID samples (3.89; 3.15–4.63) than in BN (2.43; 1.92–2.94) and BN/AID samples (2.52; 1.54–3.50) (p < 0.001). Anti-PDC-E2 avidity was higher in PBC (mean 64.5 %; 95 % CI 57.5–71.5 %) and PBC/AID samples (66.1 %; 54.4–77.8 %) than in BN samples (39.2 %; 30.9–37.5 %) (p < 0.001). PBC and PBC/AID recognized more cell domains (mitochondria, nuclear envelope, PML/sp-100 bodies, centromere) than BN (p = 0.008) and BN/AID samples (p = 0.002). Three variables were independently associated with established PBC: high-avidity anti-PDC-E2 (OR 4.121; 95 % CI 2.118–8.019); high-titer IIF-AMA (OR 4.890; 2.319–10.314); antibodies to three or more antigenic cell domains (OR 9.414; 1.924–46.060). Conclusion The autoantibody profile was quantitatively and qualitatively more robust in definite PBC as compared with AMA-positive biochemically normal individuals.

The juvenile three-spined stickleback : model organism for the study of estrogenic and androgenic endocrine disruption in laboratory and field

Industrial and domestic sewage effluents have been found to cause reproductive disorders in wild fish, often as a result of the interference of compounds in the effluents with the endocrine system. This thesis describes laboratory-based exposure experiments and a field survey that were conducted with juveniles of the three-spined stickleback, Gasterosteus aculeatus. This small teleost is a common fish in Swedish coastal waters and was chosen as an alternative to non-native test species commonly used in endocrine disruption studies, which allows the comparison of field data with results from laboratory experiments. The aim of this thesis was to elucidate 1) if genetic sex determination and differentiation can be disturbed by natural and synthetic steroid hormones and 2) whether this provides an endpoint for the detection of endocrine disruption, 3) to evaluate the applicability of specific estrogen- and androgen-inducible marker proteins in juvenile three-spined sticklebacks, 4) to investigate whether estrogenic and/or androgenic endocrine disrupting activity can be detected in effluents from Swedish pulp mills and domestic sewage treatment plants and 5) whether such activity can be detected in coastal waters receiving these effluents. Laboratory exposure experiments found juvenile three-spined sticklebacks to be sensitive to water-borne estrogenic and androgenic steroid substances. Intersex – the co-occurrence of ovarian and testicular tissue in gonads – was induced by 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 17α-methyltestosterone (MT) and 5α-dihydrotestosterone (DHT). The first two weeks after hatching was the phase of highest sensitivity. MT was ambivalent by simultaneously eliciting masculinizing and feminizing effects. When applying a DNA-based method for genetic sex identification, it was found that application of MT only during the first two weeks after hatching caused total and apparently irreversible development of testis in genetic females. E2 caused gonad type reversal from male to female. E2 and EE2 induced vitellogenin - the estrogen-responsive yolk precursor protein, while DHT and MT induced spiggin – the androgen-responsive glue protein of the stickleback. None of the effluents from two pulp mills and two domestic sewage treatment plants had any estrogenic or androgenic activity. Juvenile three-spined sticklebacks were collected during four subsequent summers at the Swedish Baltic Sea coast in recipients of effluents from pulp mills and a domestic sewage treatment plant as well as remote reference sites. No sings of endocrine disruption were observed at any site, when studying gonad development or marker proteins, except for a deviation of sex ratios at a reference site. The three-spined stickleback – with focus on the juvenile stage – was found to be a sensitive species suitable for the study of estrogenic and androgenic endocrine disruption.