Morphological and quantitative changes in paternal brood-pouch vasculature during embryonic development in two Syngnathus pipefishes.

Vascular corrosion casts of Syngnathus floridae and Syngnathus fuscus brood pouches were examined by scanning electron microscopy. Morphological and quantitative data on the vasculature of the paternal brood pouch during each stage of embryonic development were investigated to explore potential changes during brooding, to consider interspecific differences and to provide structural evidence for previously reported functional roles of the brood pouch. The brood pouches of both species are highly vascularized structures with cup-like arrangements of brood-pouch vasculature developing around each embryo shortly after fertilization and breaking down before fry release. The density and size of paternally derived blood vessels in contact with the embryos were found to be consistent for S. fuscus once this structure was established early in development. On the contrary, these vasculature measurements varied with early S. floridae brood stages when the embryo still relied heavily on the yolk sac. Diameter measurements of S. fuscus brood-pouch blood vessels were also comparatively smaller during these early developmental stages, suggesting that the structural stability and opportunity for greater transport via slower blood flow may contribute to greater paternal allocation. This is the first study to document changes in brood-pouch vasculature during specific stages of embryonic development, to show regression of this vasculature before fry release and to provide morphological data for two syngnathid species for which information on brood-pouch physiology is available.

Comparison of two sites in Mobile Bay using in vivo biomarkers in largemouth bass, sediment bioassays, and sediment contaminant analysis.

This research addresses whether chemical contaminants are having a biologically measurable effect on largemouth bass (Micropterus salmoides), an important fish species in the Mobile Bay National Estuary. Bass and sediment samples were collected in September 2001 from Big Bateau and D'Olive Bays; the latter has been suggested to be an environmentally impacted site. There was a significant difference in the age distribution of bass collected from the two sites. However, none of the animals showed any evidence of reproductive activity or had mature gametes. The age and gonadosomatic index were greater in animals collected from the Bateau site. Fish liver microsome ethoxyresorufin-O-deethylase (EROD) activities (elevated in the presence of CYP1A-inducing contaminants) were not significantly different between sites but were significantly higher in older bass. Plasma steroid concentrations in younger animals (less than 1 year old) tended to be higher and more variable than the concentrations found in older animals. Once the significant effects of age on plasma steroid concentrations were removed, an analysis of the residual change in steroids revealed no differences in testosterone or estradiol between animals collected at the two sites. Sediments were collected for chemical extraction and analysis in two bioassays: the H4IIE rat hepatoma EROD assay and the yeast estrogen screen (YES assay). The H4IIE bioassay indicated the presence of sediment BaP induction equivalents between 72 and 320 ng/g, whereas the YES assay indicated that potent estrogenic substances were not present in the sediment samples. The H4IIE bioassay results were higher for D'Olive samples, which was consistent with higher PAH concentrations in those sediments. Taken together the biomarkers and chemical analysis suggested generally low organic contamination at these two sites.

Determination of beta-adrenergic receptor blocking pharmaceuticals in United States wastewater effluent.

Beta adrenergic receptor antagonists (beta-Blockers) are frequently prescribed medications in the United States and have been identified in European municipal wastewater effluent, however no studies to date have investigated these compounds in United States wastewater effluent. Municipal wastewater effluent was collected from treatment facilities in Mississippi, Texas, and New York to investigate the occurrence of metoprolol, nadolol, and propranolol. Propranolol was identified in all wastewater samples analyzed (n = 34) at concentrations < or = 1.9 microg/l. Metoprolol and nadolol were identified in > or = 71% of the samples with concentrations of metoprolol < or = 1.2 microg/l and nadolol < or = 0.36 microg/l. Time course studies at both Mississippi plants and the Texas plant indicate that concentrations of propranolol, metoprolol, and nadolol remain relatively constant at each sampling period. This study indicates that beta-Blockers are present in United States wastewater effluent in the ng/l to microg/l range.

Toxicity of select beta adrenergic receptor-blocking pharmaceuticals (B-blockers) on aquatic organisms.

One class of pharmaceutical compounds identified in U.S. and European waters are the B-adrenergic receptor blocking compounds (B-blockers). However, little information is available on the potential aquatic toxicity of these compounds. Therefore, Hyalella azteca, Daphnia magna, Ceriodaphnia dubia, and Oryias latipes (Japanese medaka) were exposed to metoprolol, nadolol, and propranolol to determine potential toxicity. Average 48-h LC(50) for propranolol to H. azteca was 29.8 mg/L. The no-observed-effects concentration (NOEC) and lowest-observed-effects concentration (LOEC) for propranolol affecting reproduction of H. azteca were 0.001 and 0.1 mg/L, respectively. The average propranolol and metoprolol 48-h LC(50)s for D. magna were 1.6 and 63.9 mg/L, respectively. C. dubia 48-h LC(50)s were 0.85 and 8.8 mg/L for propranolol and metoprolol, respectively. The NOEC and LOEC of propranolol affecting reproduction in C. dubia were 0.125 and 0.25 mg/L, respectively. In O. latipes, the propranolol 48-h LC(50) was 24.3 mg/L. Medaka growth was decreased at 0.5 mg/L propranolol. A 2-week medaka reproductive study indicated significant changes in plasma steroid levels; however, no changes in the average number of eggs produced or number of viable eggs which hatched was observed. In a 4-week follow-up propranolol exposure, the total number of eggs produced by medaka and the number of viable eggs that hatched were decreased at concentrations as low as 0.5 microg/L. Based on this study and the expected aqueous environmental exposure levels, adverse effects of propranolol to invertebrate populations is unlikely; however, further reproductive studies are need to elucidate the risk to teleosts.

Exposure to environmentally relevant concentrations of different nonylphenol formulations in Japanese medaka.

The time course of exposure to p-nonylphenol (NP) from two different sources was compared to equivalent exposures of 17-beta-estradiol (E2) and a solvent control (ethanol; EtOH). Japanese medaka were exposed for 4 days to a nominal concentration of 20 micrograms/l of either NP-I (Schenectady International, Inc.), NP-II (Aldrich), or E2, and were then placed in untreated water for 5 days. Tissue samples were taken at two time points during the 4-day exposure and two time points during the 5 days following exposure. Liver homogenates were analyzed using a western blot to detect vitellogenin (VTG) and quantified by measuring the optical density for each lane. Preliminary results indicate that E2 significantly increased VTG staining above the level observed in EtOH-treated controls for both males and females. A two-way analysis of variance (ANOVA) indicates that NP from both sources, as well as E2, significantly increased VTG staining in males (ANOVA, n = 48, P < 0.001; Tukey pairwise tests, all P < 0.008). A significant increase in VTG was observed in E2-treated males and females the first day following transfer into toxicant-free water (two-way ANOVAs, both n = 48, P < 0.003; Tukey pairwise tests, all P < 0.019). If confirmed, this extended response observed for low-level exposures may represent a significant factor for sampling scenarios following pulsitile exposure.

Developmental evaluation of a potential non-steroidal estrogen: triclosan.

Triclosan is an antibacterial agent commonly used in industry and often detected in waste-water effluent. The potential of triclosan to act as an endocrine disruptor was examined because its chemical structure closely resembles known non-steroidal estrogens (e.g. DES, bisphenol A). Japanese medaka fry (Oryzias latipes) were exposed for 14 days beginning 2 days post-hatch to triclosan (100, 10, 1 micrograms/l), 17-beta estradiol (E2; 1 microgram/l), or a solvent control (ethanol). Two months post-exposure, the phenotypic sex of each adult was assessed visually using sexually dimorphic fin shape and size. The proportion of females in each group was similar for triclosan-exposed animals and solvent-treated controls (ethanol 53%, 1 ppb 58%, 10 ppb 45%, 100 ppb 36%) although E2 treatment did produce 92% female adults. Sexually dimorphic fin traits were quantified to look for potential effects of triclosan and E2 on the development of secondary sexual characters. These results do not support the hypothesis that triclosan is potently estrogenic. However, changes in fin length and non-significant trends in sex ratio suggest triclosan is potentially weakly androgenic.

Preoptic GnRH and AVT: axes for sexual plasticity in teleost fish.

Alternative reproductive tactics within one sex, adult sex or role change, and reproductive suppression are all forms of reproductive plasticity commonly exhibited among teleost fishes. The two neuropeptides that have been most extensively studied with regard to such behavioral plasticity are gonadotropin releasing hormone (GnRH) and arginine vasotocin (AVT). Here, we review intra- and intersexual variation in the number and size of GnRH and AVT neurons along with gonadal phenotype in those species of teleosts showing intraspecific plasticity in reproductive behavior. In several species, male dimorphisms in the number and/or size of GnRH neurons in the forebrain's preoptic area parallel a divergence in relative gonad size and reproductive tactics. The available studies of AVT-containing neurons in the preoptic area also indicate intrasexual dimorphisms among males, although a proximate link to other reproductive traits and behavioral outcomes is more difficult to recognize. For both GnRH and AVT, there are also species-typical patterns in the coupling between structural (e.g., neuronal and gonadal) traits and reproductive tactic expressed, which likely reflect distinct patterns of adaptation to particular ecological environments. As discussed, neurophysiological, biochemical, and receptor density studies are now essential to establish the functional significance of the diverse organizational patterns of GnRH and AVT neurons in teleosts. Similar studies also need to be carried out in species of other vertebrate groups that show comparable behavioral plasticity.

Preoptic AVT immunoreactive neurons of a teleost fish with alternative reproductive tactics.

Recent reports have implicated an important role for arginine vasotocin (AVT) in the socially mediated sexual differentiation of fishes. This study focuses on the plainfin midshipman (Porichthys notatus) which exhibits two male morphs, type I and type II, differing in a suite of behavioral, neurobiological, and endocrine traits. Immunocytochemical techniques were used to detect neurons containing AVT-like peptide in the forebrain of juveniles, adult females, and type I and type II males. AVT immunoreactive (ir) somata were localized to three regions: the terminal nerve ganglion, the preoptic area (POA), and the pineal stalk. The profile area, or size, of AVT-ir POA neurons differed across the four classes of midshipman and was strongly correlated to differences in body size among the groups. By contrast, the number of AVT-ir cells in the POA exhibited no difference across the classes of midshipman. The number of POA cells containing AVT is therefore likely to be set early in development and not to change with the growth of the animal. An analysis of AVT-ir cell number normalized by body mass revealed that the larger morphs, type I males and females, have fewer cells per gram body mass than type II males and juveniles. Therefore, type II males have a juvenile-like AVT POA phenotype with smaller cells and more numerous cells per unit body mass than type I males. Type II males also exhibit more variability in the number of AVT-ir cells found in the POA compared to type I males.

Modification of gonadotropin releasing hormone (GnRH) mRNA expression in the retinal-recipient Thalamus.

Although the environmental cues that trigger reproductive behaviors are known for many species, the mechanisms through which these signals influence the neurochemistry of the brain to produce behavior have been elusive. In this study, we describe a retinally modulated system of gonadotropin releasing hormone (GnRH) producing neurons in the thalamus of the plainfin midshipman fish, Porichthys notatus. Previously, we cloned and sequenced the cDNA for prepro-GnRH in midshipman. Here, using in situ hybridization, we localized prepro-GnRH mRNA to the ventrolateral nucleus of the thalamus, three divisions of the preoptic area, the ganglion of the terminal nerve, and the olfactory bulb. Since the thalamus, terminal nerve ganglion, and preoptic area have been associated with visual functions, we investigated the retinal connections in midshipman. In particular, biocytin tract tracing delineated a reciprocal connection between the ventrolateral nucleus of the thalamus and the retina. Retinofugal projections are exclusively contralateral. Experimental manipulation of this retinalthalamic loop through complete optic nerve transection shows that GnRH mRNA expression in the contralateral ventrolateral nucleus may be influenced by the retina. We hypothesize that a reciprocal retinothalamic GnRH circuit is important in modulating the expression of seasonal reproductive behaviors.

Head entrapment and neonatal outcome by mode of delivery in breech deliveries from 28 to 36 weeks of gestation.

OBJECTIVES: We attempted to determine whether there are differences in the incidence of head entrapment and adverse neonatal outcome by mode of delivery in breech deliveries from 28 to 36 weeks of gestation. STUDY DESIGN: Computerized data and charts of 321 viable consecutive singleton breech deliveries from 28 to 36 weeks' gestation were reviewed. Statistical methods used included chi2, logistic regression, and analysis of variance. RESULTS: Head entrapment occurred in 4 of 52 (7.7%) neonates delivered vaginally and 14 of 269 (5.2%) neonates delivered by cesarean section (p = 0.48). There were no statistically significant associations between head entrapment and adverse neonatal outcomes. CONCLUSIONS: There was no significant difference in the incidence of head entrapment by mode of delivery for breech infants at 28 to 36 weeks' gestation, nor was there an association with adverse neonatal outcomes after entrapment.

Head entrapment and neonatal outcome by mode of delivery in breech deliveries from twenty-four to twenty-seven weeks of gestation.

OBJECTIVES: Our purpose was to determine whether there are differences in the incidence of head entrapment and adverse neonatal outcome by mode of delivery in breech deliveries from 24 to 27 weeks of gestation. STUDY DESIGN: Charts of 132 viable consecutive singleton breech deliveries from 24 to 27 weeks' gestation were reviewed. Statistical methods used included chi 2, logistic regression, and analysis of variance. RESULTS: Head entrapment occurred in four of 43 (9.3%) vaginally delivered neonates and five of 89 (5.6%) neonates delivered by cesarean section (p = 0.17). The only identifiable risk factor for head entrapment was the birth weight category 1000 to 1249 gm (odds ratio 6.4, 95% confidence interval 1.6 to 26.1). There were no statistically significant associations between head entrapment and adverse neonatal outcomes. CONCLUSIONS: There did not appear to be a difference in the incidence of head entrapment by mode of delivery for breech infants at 24 to 27 weeks' gestation, nor did there appear to be a difference in adverse neonatal outcomes after entrapment. However, this latter conclusion has limited power because of the small number of head entrapment cases.