Single and mixture per- and polyfluoroalkyl substances accumulate in developing Northern leopard frog brains and produce complex neurotransmission alterations.

Per- and polyfluoroalkyl substances (PFAS) are present in water and >99% of human serum. They are found in brains of wildlife; however, little is known about effects on the developing brain. To determine the effects of PFAS on brain and cardiac innervation, we conducted an outdoor mesocosm experiment with Northern leopard frog larvae (Rana pipiens) exposed to control, 10 ppb perfluorooctane sulfonate (PFOS), or a PFAS mixture totaling 10 ppb that mimicked aqueous film forming foam-impacted surface water (4 ppb PFOS, 3 ppb perfluorohexane sulfonate, 1.25 ppb perfluorooctanoate, 1.25 ppb perfluorohexanoate, and 0.5 ppb perfluoro-n-pentanoate). Water was spiked with PFAS and 25 larvae (Gosner stage (GS) 25) added to each mesocosm (n = 4 mesocosms per treatment). After 30 days, we harvested eight brains per mesocosm and remaining larvae developed to GS 46 (i.e. metamorphosis) before brains and hearts were collected. Weight, length, GS, and time to metamorphosis were recorded. Brain concentrations of all five PFAS were quantified using LC/MS/MS. Dopamine and metabolites, serotonin and its metabolite, norepinephrine, γ-aminobutyric acid, and glutamate were quantified using High Performance Liquid Chromatography with electrochemical detection while acetylcholine and acetylcholinesterase activity were quantified with the Invitrogen Amplex Red Acetylcholine Assay. PFOS accumulated in the brain time- and dose-dependently. After 30 days, the mixture decreased serotonin while both PFAS treatments decreased glutamate. Interestingly, acetylcholine increased in PFAS treatments at GS 46. This research shows that developmental environmentally relevant exposure to PFAS changes neurotransmitters, especially acetylcholine.

Lipid dynamics in zebrafish embryonic development observed by DESI-MS imaging and nanoelectrospray-MS.

The zebrafish Danio rerio is a model vertebrate organism for understanding biological mechanisms. Recent studies have explored using zebrafish as a model for lipid-related diseases, for in vivo fish bioassays, and for embryonic toxicity experiments. Mass spectrometry (MS) and MS imaging are established tools for lipid profiling and spatial mapping of biomolecules and offer rapid, sensitive, and simple analytical protocols for zebrafish analysis. When ambient ionization techniques are used, ions are generated in native environmental conditions, requiring neither sample preparation nor separation of molecules prior to MS. We used two direct MS techniques to describe the dynamics of the lipid profile during zebrafish embryonic development from 0 to 96 hours post-fertilization and to explore these analytical approaches as molecular diagnostic assays. Desorption electrospray ionization (DESI) MS imaging followed by nanoelectrospray (nESI) MS and tandem MS (MS/MS) were used in positive and negative ion modes, allowing the detection of a large variety of phosphatidylglycerols, phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols, ubiquinone, squalene, and other lipids, and revealed information on the spatial distributions of lipids within the embryo and on lipid molecular structure. Differences were observed in the relative ion abundances of free fatty acids, triacylglycerols, and ubiquinone - essentially localized to the yolk - across developmental stages, whereas no relevant differences were found in the distribution of complex membrane glycerophospholipids, indicating conserved lipid constitution. Embryos exposed to trichloroethylene for 72 hours exhibited an altered lipid profile, indicating the potential utility of this technique for testing the effects of environmental contaminants.

Antagonists to the Wnt cascade exhibit sex-specific expression in gonads of sexually mature shovelnose sturgeon.

Little is known regarding molecular mechanisms involved in sex determination and differentiation in sturgeon species. We addressed this knowledge gap by using next generation pyrosequencing technology to provide transcript libraries and species-specific sequences for mature gonads of shovelnose sturgeon, Scaphirhynchus platorynchus. We then mined these libraries to identify gender-specific transcripts and quantified relative transcript abundance using quantitative real-time polymerase chain reaction (qPCR). We detected a limited number of genes known to play a role in sex differentiation in other species. The sequence for dmrt1 was found only in the testes library. The abundance of dmrt1 differed slightly between the sexes, but the melt curve suggests that there may be different isoforms of dmrt1 in ovaries and testes of shovelnose sturgeon. The transcription factor foxl2 was 5.3-fold greater in ovaries than in testes. Two antagonists to the Wnt cascade, dickkopf-1 (dkk1) and dapper-1 (dact1), were found only in the ovary library. Results from qPCR indicated that dkk1 and dact1 were upregulated 1,819.1- and 207.5-fold, respectively, in ovaries compared with testes. These results suggest that antagonists to the Wnt cascade may play significant roles in sex differentiation and gonadal development in sturgeon and could serve as sex markers in this group of ancient fish.

Sex-specific gonadal and gene expression changes throughout development in fathead minnow.

Although fathead minnows (Pimephales promelas) are commonly used as a model fish in endocrine disruption studies, past studies have not characterized sex-specific baseline expression of genes involved in sex differentiation during development in this species. Using a sex-linked DNA marker to verify gender, we evaluated the expression over time of genes involved in sex differentiation (dmrt1, cyp19a, cyp17, star, esr1, ar) in developing fathead minnows (10-45 days post hatch). Evaluation of these molecular markers in combination with gender identification help us to better understand the mechanisms regulating sex differentiation in fathead minnows and how endocrine-disrupting chemicals may alter these processes.

Helminth parasites of eastern box turtles (Terrapene carolina carolina) from southern Indiana, USA.

Very little is known about parasitic diseases of eastern box turtles (Terrapene carolina carolina). The objective of this study was to examine the parasitic fauna of eastern box turtles collected from southern Indiana, USA. Turtles (n = 40) were salvaged mostly as road kills from southern Indiana between May and October 2009. Seven species of helminths in total were found parasitizing the gastrointestinal tract, including two digenean trematodes (Brachycoelium salamandrae and Telorchis robustus) and five nematodes (Oswaldocruzia pipiens, Cosmocercoides dukae, Falcaustra affinis, F. chelydrae and Serpinema trispinosus). We report prevalence, abundance and mean intensity of infection for all helminths. Helminths were not found in any other organs examined (heart, gonads, liver, heart, kidney and urinary bladder) and no ectoparasites were found. Overall, mean intensity of infections was low (1-14 parasites/host), suggesting that these parasites are unlikely to be associated with negative health impacts. This constitutes the first study of this kind for Indiana.

An evaluation of the effects of persistent environmental contaminants on the reproductive success of Great Blue Herons (Ardea herodias) in Indiana.

Contaminants in Great Blue Herons (Ardea herodias) from Indiana were quantified to determine if levels were high enough to impair reproduction. During 2005 and 2006, 35 eggs were collected from 6 colonies and analyzed for contaminants. Between 30 and 101 nests were monitored in 7 colonies weekly over a 3-month period to determine reproductive and fledging success. Average levels (+/-SD) of polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and organochlorine pesticides in egg yolks were 3,101 (+/-4,737), 7.20 (+/-2.96), and 2,869 (+/-2,291) ppb, respectively. Reproductive success (average number of chicks fledged per active nest) and fledging success (number of chicks fledged per successful nest) averaged 1.52 and 1.92 chicks, respectively. Contaminant levels measured in eggs from this region are comparable to those observed not having affects on reproductive success elsewhere; therefore, factors other than environmental contamination may be affecting reproductive success of Great Blue Herons in study colonies.

Mercury in feathers from Chilean birds: influence of location, feeding strategy, and taxonomic affiliation.

This study reports baseline concentrations of mercury (Hg) in feathers from different species of birds sampled at various locations off the Chilean coast (Southeastern Pacific). Hg concentrations were evaluated in relation to geographic location, taxonomic affiliation, and feeding strategies. Between January and March of 1995, we collected mature contour feathers from 116 birds belonging to 22 species, mostly seabirds. Birds were collected from 10 different locations (26 degrees 09'S, 70 degrees 40'W to 54 degrees 56'S, 67 degrees 37'W). Feather Hg concentrations ranged from 0.11 to 13 microg g(-1) dry weight. We found differences in feather Hg concentrations across taxonomic groups, with highest concentrations in petrels, shearwaters, and albatrosses (Procellaridae), followed by boobies (Sulidae), gulls, terns, skuas (Laridae) and cormorants (Phalacrocoracidae). Ibises and oystercatchers (Threskiornithidae and Charadriidae) had intermediate values, whereas ducks and geese (Anatidae) contained the least amount of Hg. Oceanic species preying on mesopelagic fish (the Procellariformes albatrosses, petrels, and fulmars) had over twice as much Hg (overall average of 3.9 microg g(-1)) when compared to the rest of the species sampled (overall average of 1.5 microg g(-1)). We did not find higher Hg concentrations in birds inhabiting the more heavily industrialized and urbanized areas of the country (central and northern regions), but in birds inhabiting the remote Juan Fernandez Archipelago. This is not surprising, since all the Procellariformes (the group with highest Hg values in this study) were collected from these islands. Except for Hg in Kermadec petrels (mean of 12 microg g(-1)), the range of Hg values reported here (0.11-7.3 microg g(-1)) fell below those known to cause adverse health and reproductive effects in birds.

Assessment of reproductive effects in largemouth bass (Micropterus salmoides) exposed to bleached/unbleached kraft mill effluents.

This study evaluated the potential effects of different concentrations of bleached/unbleached kraft mill effluent (B/UKME) on several reproductive endpoints in adult largemouth bass (Micropterus salmoides). The kraft mill studied produces a 50/50 mix of bleached/unbleached market pulp with an estimated release of 36 million gal of effluent/day. Bleaching sequences were C90d10EopHDp and CEHD for softwood (pines) and hardwoods (mainly tupelo, gums, magnolia, and water oaks), respectively. Bass were exposed to different effluent concentrations (0 [controls, exposed to well water], 10, 20, 40, or 80%) for either 28 or 56 days. At the end of each exposure period, fish were euthanized, gonads collected for histological evaluation and determination of gonadosomatic index (GSI), and plasma was analyzed for 17beta-estradiol, 11-ketotestosterone, and vitellogenin (VTG). Largemouth bass exposed to B/UKME responded with changes at the biochemical level (decline in sex steroids in both sexes and VTG in females) that were usually translated into tissue/organ-level responses (declines in GSI in both sexes and in ovarian development in females). Although most of these responses occurred after exposing fish to 40% B/UKME concentrations or greater, some were observed after exposures to 20% B/UKME. These threshold concentrations fall within the 60% average yearly concentration of effluent that exists in the stream near the point of discharge (Rice Creek), but are above the <10% effluent concentration present in the St. Johns River. The chemical(s) responsible for such changes as well as their mode(s) of action remain unknown at this time.

Results of assisted reproduction techniques in Latin America.

The fact that today it is possible to write a comprehensive report on assisted reproduction technologies in Latin America is the result of a serious and systematic effort, accomplished by more than 80 centres, from Mexico to Chile. Over the past 10 years, these centres representing the vast majority of assisted reproductive treatment cycles performed in Latin America, have agreed voluntarily to report their work to our regional registry using a pre-established format. Furthermore, during 1999 and part of 2000, all centres were visited by a team of biologists and clinicians, who evaluated for consistencies in the data reported. This activity also included an evaluation of personnel, facilities, etc. It can be proudly said that the data reported are a true reflection of the actual situation in our region.

Prospective randomized trial to evaluate the efficacy of a vaginal ring releasing progesterone for IVF and oocyte donation.

A polysyloxane vaginal ring containing 1g of natural progesterone was developed as luteal supplementation for women treated with IVF-embryo transfer and for agonadal women participating in an oocyte donation programme. The ring provides continuous release of progesterone (10-20 nmol/l) for 90 days. The efficacy of this form of progesterone supplementation was evaluated in two multicentre prospective randomized trials. IVF-embryo transfer trial: After oocyte aspiration, 505 women were randomly allocated to progesterone supplementation with vaginal ring or i.m. progesterone (50 mg/day). The clinical pregnancy rate was 36.6% in both groups. Implantation rate was 15.9% in the vaginal ring and 16.0% in i.m. progesterone. Oocyte donation trial: After endometrial proliferation with micronized oestradiol, 153 women were allocated to progesterone replacement with a vaginal ring or i.m. progesterone (100 mg/day). Clinical pregnancy rate was 39.8 and 28.6% respectively. Implantation rate was significantly higher with the vaginal ring compared with i.m. progesterone (19.9 and 11.6% respectively, P = 0.006). The vaginal ring is a novel development which provides continuous release of progesterone for 90 days. In IVF-embryo transfer, its effectiveness is similar to daily i.m. injections. In oocyte donation the ring provides a progestative milieu which improves the implantation rate and eliminates the discomfort of daily i.m. injections.

Effects of mercury on health and first-year survival of free-ranging great egrets (Ardea albus) from southern Florida.

The objectives of this study were to determine whether elevated mercury (Hg) concentrations have a negative impact on the health and survival of nestling and juvenile free-ranging great egrets (Ardea albus) from southern Florida. During 1994, when health and survival was monitored in a cohort of young birds with naturally variable concentrations of Hg, packed cell volume was positively correlated with blood Hg concentrations, and high Hg concentration in blood was not related to the probability of surviving during the first 10.5 months of life. During 1995, 70 first-hatched great egret chicks were included in a Hg field-dosing experiment to compare the effects of elevated Hg on health and survival. Birds were dosed while in the nest orally every 2.5 days for 15 days with 0.5 mg of methyl mercury chloride (MeHgCl) for an estimated intake of 1.54 mg MeHgCl/kg food intake. These birds were compared with controls, which received an estimated 0.41 mg MeHgCl/kg food. No differences were observed in health parameters or in the probability of surviving during the first 8 months of age between egrets that were dosed with Hg and those that were not. A likely explanation for the lack of any effects on health and survival between both groups could be that chicks at this age were eliminating most of the dietary Hg through the production of new feathers.

The polysulphate binding domain of human proacrosin/acrosin is involved in both the enzyme activation and spermatozoa-zona pellucida interaction.

Mammalian acrosin is a protease present as a zymogen in the acrosome of a non-reacted mammalian sperm, and in vitro is able to carry out limited hydrolysis of homologous and heterologous zonae pellucidae. On the other hand, sulphated polymers and zona pellucida glycoproteins bind to acrosin on a domain different from the active site, named the polysulphate binding domain (PSBD). Thus it is believed that acrosome-reacted spermatozoa bind to glycan chains of the zona pellucida through PSBD participating as secondary binding receptor. The aim of the present work was to study the role of PSBD during both human gamete interaction and acrosin activation. In this work we present evidence that the anti-human acrosin monoclonal antibody C5F10 is directed to an epitope located on or near the PSBD on human proacrosin/acrosin. Moreover, we show that this antibody is able to inhibit both proacrosin activation induced by fucoidan and the sperm binding to the zona pellucida. Our results suggest that the same PSBD is involved in both sperm secondary binding, during zona pellucida penetration, and proacrosin activation.

Hookworms (Uncinaria sp.) in Juan Fernandez fur seal pups (Arctocephalus philippii) from Alejandro Selkirk Island, Chile.

Sixty Juan Fernandez fur seal pups (Arctocephalus philippii) found dead on Alejandro Selkirk Island, Chile, during the breeding seasons of 1991 and 1992 were examined for the presence of hookworms (Uncinaria sp.). Hookworms were found in 60% of the pups examined, at a mean intensity of 17 parasites per infected host. Intensity of infection did not differ among years or sexes. There was a significant positive relationship between thickness of subcutaneous fat and number of hookworms.

New records of feather lice from some seabirds in Chile.

Six species of chewing lice (Phthiraptera: Menoponidae, Philopteridae) are reported from four species of seabirds collected off the coast of Chile. They are Eidmanniella pellucida, Piagetiella transitans, and Piagetiella caputincisa, from a Guanay cormorant (Phalacrocorax bougainvillii); Harrisoniella ferox collected from a black-browed albatross (Diomedea melanophrys); Ancistrona vagelli from a white-necked petrel (Pterodroma externa); and Pelmatocerandra flinti collected from a Magellan diving petrel (Pelecanoides magellani). Eidmanniella pellucida and P. transitans are reported from Chile for the first time. Piagetiella caputincisa from the Guanay cormorant may be a contaminant from a red-legged shag (Phalacrocorax gaimardi).

Immobilization of Juan Fernandez fur seals, Arctocephalus philippii, with ketamine hydrochloride and diazepam.

During November and December of 1988, 1990, and 1991, a total of 22 free-ranging Juan Fernández fur seal (Arctocephalus philippii) females from Alejandro Selkirk Island, Juan Fernández Archipelago, Chile, were immobilized with a combination of ketamine and diazepam. Atropine sulphate was used to decrease respiratory secretions. The mean (+/- SD) induction dosages of ketamine and diazepam were 3.64 +/- 1.3 mg/kg and 0.12 +/- 0.07 mg/kg, respectively. Mean (+/- SD) induction time and time to recovery for females injected intramuscularly (IM) (15 +/- 7 min and 47 +/- 16 min) were significantly greater than for females injected intravenously (IV) (0.6 +/- 0.4 min and 26 +/- 11 min). Mean (+/- SD) heart rates and core temperatures were significantly higher for females injected IV (173 +/- 15.71 beats/min and 37.6 +/- 0.83 C) than for females injected IM (135 +/- 27.06 beats/min and 36.5 +/- 1.15 C). In addition, the IV route resulted in better levels of immobilization compared to the IM route. The degree of immobilization was not related to the dosages of ketamine and diazepam administered. Two animals died after drug administration.

Inhibitory effect of a Microcystis sp (cyanobacteria) toxin on development of preimplantation mouse embryos.

1. A soluble toxin, purified from the algae bloom of an eutrophic lake dominated by Microcystis, is a very effective inhibitor of early embryo development in a dose-response relationship. 2. Two- and 8-cell mouse embryos under the influence of Microcystis toxin do not reach the developmental stages of morula and blastocyst, respectively. 3. Actin cortex is disorganized without change in the microtubules structure. 4. Results are discussed in terms of the possible mechanisms by which the toxin arrests development considering, specifically, effects on the cytoskeleton and/or on voltage-insensitive transmembrane Ca2+ channels.