Amphibian Assisted Reproductive Technologies: Moving from Technology to Application.

Amphibians have experienced a catastrophic decline since the 1980s driven by disease, habitat loss, and impacts of invasive species and face ongoing threats from climate change. About 40% of extant amphibians are under threat of extinction and about 200 species have disappeared completely. Reproductive technologies and biobanking of cryopreserved materials offer technologies that could increase the efficiency and effectiveness of conservation programs involving management of captive breeding and wild populations through reduced costs, better genetic management and reduced risk of species extinctions. However, there are relatively few examples of applications of these technologies in practice in on-the-ground conservation programs, and no example that we know of where genetic diversity has been restored to a threatened amphibian species in captive breeding or in wild populations using cryopreserved genetic material. This gap in the application of technology to conservation programs needs to be addressed if assisted reproductive technologies (ARTs) and biobanking are to realise their potential in amphibian conservation. We review successful technologies including non-invasive gamete collection, IVF and sperm cryopreservation that work well enough to be applied to many current conservation programs. We consider new advances in technology (vitrification and laser warming) of cryopreservation of aquatic embryos of fish and some marine invertebrates that may help us to overcome factors limiting amphibian oocyte and embryo cryopreservation. Finally, we address two case studies that illustrate the urgent need and the opportunity to implement immediately ARTs, cryopreservation and biobanking to amphibian conservation. These are (1) managing the biosecurity (disease risk) of the frogs of New Guinea which are currently free of chytridiomycosis, but are at high risk (2) the Sehuencas water frog of Bolivia, which until recently had only one known surviving male.

Radial glial cell: critical functions and new perspective as a steroid synthetic cell.

The radial glial cell (RGC) is a glial cell type in the central nervous system of all vertebrates. Adult teleost fish have abundant RGCs in the brain in contrast to mammals. Adult fish RGCs have many important functions, including forming a structural scaffold to guide neuronal migration and serving as the progenitor cells in the brain to generate neurons. The role of the RGC in adult neurogenesis explains the high regenerative capacity of adult fish brain. There is increasing evidence from several species that some glial cells produce or metabolize steroids. It is now well-known that teleost RGCs express aromatase and produce estrogens from androgen precursors, which may be important for local neuroendocrine functions and regulation of neurogenesis. The question of whether RGCs are capable of de novo steroid synthesis from cholesterol remains unanswered. However, the expression of steroidogenic acute regulatory protein, and the key enzyme cytochrome P450 17alpha-hydroxylase in primary cultures of goldfish RGCs indicate the potential to produce 17α-hydroxy-pregnenolone and thus other steroid intermediates. The possibility of synthesizing additional non-estrogenic steroids may indicate new functions for the RGC.

Naphthenic acid fraction compounds reduce the reproductive success of wood frogs (Rana sylvatica) by affecting offspring viability.

Understanding the toxicity of organic compounds in oil sands process-affected water (OSPW) is necessary to inform the development of environmental guidelines related to wastewater management in Canada's oil sands region. In the present study, we investigated the effects of naphthenic acid fraction compounds (NAFCs), one of the most toxic components of OSPW, on mating behaviour, fertility, and offspring viability in the wood frog (Rana sylvatica). Wild adult wood frogs were exposed separately from the opposite sex to 0, 5, or 10 mg/L of OSPW-derived NAFCs for 24 h and then combined in outdoor lake water mesocosms containing the same NAFC concentrations (n = 2 males and 1 female per mesocosm, n = 3 mesocosms per treatment). Mating events were recorded for 48 h and egg masses were measured to determine adult fertility. NAFC exposure had no significant effect on mating behaviour (probability of amplexus and oviposition, amplexus and oviposition latency, total duration of amplexus and number of amplectic events) or fertility (fertilization success and clutch size). Tadpoles (50 individuals per mesocosm at hatching, and 15 individuals per mesocosm from 42 d post-hatch) were reared in the same mesocosms under chronic NAFC exposure until metamorphic climax (61-85 d after hatching). Offspring exposed to 10 mg/L NAFCs during development were less likely to survive and complete metamorphosis, grew at a reduced rate, and displayed more frequent morphological abnormalities. These abnormalities included limb anomalies at metamorphosis, described for the first time after NAFC exposure. The results of this study suggest that NAFCs reduce wood frog reproductive success through declines in offspring viability and therefore raise the concern that exposure to NAFCs during reproduction and development may affect the recruitment of native amphibian populations in the oil sands region.

Estradiol and triiodothyronine differentially modulate reproductive and thyroidal genes in male goldfish.

While the reproductive and thyroidal systems are extensively studied in fish, they are largely studied in isolation from one another, but there is evidence supporting cross-regulation between these two systems. To better understand hormone action and the potential cross-regulation between estrogen and thyroid hormones, we examined gene expression changes in estrogen receptor (ER) and thyroid receptor (TR) subtypes and key enzymes responsible for the local synthesis and availability of estrogen and thyroid hormones (aromatase B and deiodinase, respectively) in sexually regressed, adult, male goldfish in response to 3 days waterborne exposures to 17ß-estradiol (E2; 1 nM), triiodothyronine (T3; 20 and 100 nM), and co-treatments thereof. Treatments with E2 alone did not effect ER subtype transcripts in the liver, telencephalon, or testis; however, in the testis, 1 nM T3 decreased ERα and ERß1 and co-treatments of T3 and E2 decreased ERß1 levels. TRα-1 and TRß transcripts were not auto-regulated by T3 or cross-regulated by E2. Although deiodinase type I levels were also unaffected, deiodinase type II decreased in response to T3 treatments. Liver deiodinase type III transcripts increased in response to T3 treatments, while E2 exhibited antagonistic effects on this T3-mediated induction. These results provide novel evidence of cross-talk between the reproductive and thyroid endocrine axes in a model teleost.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life-history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967-1977. © 2019 SETAC.

Toxicokinetics and bioaccumulation of polycyclic aromatic compounds in wood frog tadpoles (Lithobates sylvaticus) exposed to Athabasca oil sands sediment.

We performed accumulation-elimination experiments of polycyclic aromatic compounds (PACs) in wood frog tadpoles (Lithobates sylvaticus) using river sediment from Canada's Athabasca oil sands region. The PACs in wood frog tadpoles were ∼2x higher on average when the animals were in direct contact with PAC-contaminated sediment than when they were separated from the sediment with a screen and exposed only to aqueous PACs. These results suggest that sediment exposure/ingestion contributes as much to PAC accumulation in tadpoles as exposure via aqueous pathways. Alkyl-substituted PAC concentrations in exposed tadpoles exceeded those of the unsubstituted (parent) PACs by about 10 × . Bioaccumulation factors ranged between 0.01 and 4.93, with parent PACs having higher bioaccumulation factors than alkylated PACs. Wood frog tadpoles efficiently eliminated and metabolized most parent and alkyl-substituted PACs, though some compounds (e.g., C4-naphthalenes) had higher bioaccumulation potential and may serve as effective markers of exposure. Here we present a comprehensive analysis of the toxicokinetics and bioaccumulation of PACs (52 analytes) in amphibian larvae, and highlight the importance of sediment exposure when considering the bioaccumulation and potential biological impact of PACs in benthic and epibenthic organisms.

Auto-regulation of estrogen receptor subtypes and gene expression profiling of 17beta-estradiol action in the neuroendocrine axis of male goldfish.

Auto-regulation of the three goldfish estrogen receptor (ER) subtypes was examined simultaneously in multiple tissues, in relation to mRNA levels of liver vitellogenin (VTG) and brain transcripts. Male goldfish were implanted with a silastic implant containing either no steroid or 17beta-estradiol (E2) (100 microg/g body mass) for one and seven days. Liver transcript levels of ERalpha were the most highly up-regulated of the ERs, and a parallel induction of liver VTG was observed. In the testes (7d) and telencephalon (7d), E2 induced ERalpha. In the liver (1d) and hypothalamus (7d) ERbeta1 was down-regulated, while ERbeta2 remained unchanged under all conditions. Although aromatase B levels increased in the brain, the majority of candidate genes identified by microarray in the hypothalamus (1d) decreased. These results demonstrate that ER subtypes are differentially regulated by E2, and several brain transcripts decrease upon short-term elevation of circulating E2 levels.

Tadpoles of the horned frog Ceratophrys ornata exhibit high sensitivity to chlorpyrifos for conventional ecotoxicological and novel bioacoustic variables.

Previous studies reported that some species of the family Ceratophryidae are able to produce sounds during premetamorphic tadpole stages. We have now determined the effects of the cholinesterase-inhibiting insecticide chlorpyrifos (CPF) on sounds emitted by tadpoles of Ceratophrys ornata. Tadpoles were exposed individually in order to evaluate the progression of effects. Effects on sound production were complemented with common ecotoxicological endpoints (mortality, behavior, abnormalities and growth inhibition). C. ornata was found to be more sensitive than other native (= 67%, 50%) and non-native species (= 75%, 100%) considering lethal and sublethal endpoints, respectively. Effects on sounds appear along with alterations in swimming, followed by the presence of mild, then severe abnormalities and finally death. Therefore, sound production may be a good biomarker since it anticipates other endpoints that are also affected by CPF. Ceratophrys ornata is a promising new model species in ecotoxicology.

The effects of GABA agonists on glutamic acid decarboxylase, GABA-transaminase, activin, salmon gonadotrophin-releasing hormone and tyrosine hydroxylase mRNA in the goldfish (Carassius auratus) neuroendocrine brain.

GABA plays a pivotal role in reproduction by regulating luteinising hormone (LH) release from the anterior pituitary. Current evidence indicates that there is a prominent stimulatory effect of GABA on LH release in teleost fish which results from enhanced gonadotrophin-releasing hormone (GnRH) release and decreased dopamine turnover in the brain and pituitary. We hypothesised that there may be additional mechanisms underlying LH release in goldfish and investigated the relative mRNA levels of GABA synthesising enzymes (GAD65 and GAD67), degrading enzyme (GABA-T), activin betaa and betab, salmon GnRH (sGnRH), and tyrosine hydroxylase (TH) with the real-time reverse transcriptase-polymerase chain reaction after GABA agonist treatment. Sexually regressed female goldfish were i.p. injected with either the GABA(A) agonist muscimol (1 microg/g body weight) or the GABA(B) agonist baclofen (10 microg/g body weight). Both agonists significantly increased serum LH after 6 h. Muscimol decreased GAD65 (approximately ten-fold), GABA-T (approximately 15-fold) and TH (approximately three-fold) mRNA in the telencephalon. Baclofen significantly reduced GAD67 (approximately two-fold) and GABA-T (approximately two-fold) mRNA levels in the hypothalamus. Activin betaa, but not activin betab, steady-state mRNA was increased approximately three- to four-fold in both the hypothalamus and telencephalon after baclofen treatment. There was no change in sGnRH mRNA levels in either tissue after GABA agonist treatment. We show that the GABA(A) and GABA(B) receptor agonists have differing and rapid effects on gene transcription in the goldfish neuroendocrine brain and, by affecting specific targets, we identify putative genomic mechanisms underlying GABA-stimulated LH release in fish.

Investigation of pathogenic Escherichia coli and microbial pathogens in pulp and paper mill biosolids.

Biosolids produced from pulp and paper mill wastewater treatment have excellent properties as soil conditioners, but often contain high levels of Escherichia coli. E. coli are commonly used as indicators of fecal contamination and health hazard; therefore, their presence in biosolids causes concern and has lead to restrictions in land-spreading. The objectives of this study were to determine the following: (1) if E. coli from the biosolids of a wastewater-free pulp and paper mill were enteric pathogens, and (2) if other waterborne microbial pathogens were present. E. coli were screened for heat-labile and heat-stable enterotoxin and verocytotoxin virulence genes using a polymerase chain reaction. Ten isolates were also screened for invasion-associated locus and invasion plasmid antigen H genes. None of the 120 isolates carried these genes. Tests for seven other microbial pathogens were negative. Effluents and biosolids from this mill do not contain common microbial pathogens and are unlikely to pose a health hazard.

Mortality, morbidity and healthcare expenditures after local tumour ablation or partial nephrectomy for T1A kidney cancer.

BACKGROUND: Local tumour ablation (LTA) may yield better perioperative outcomes than partial nephrectomy (PN), however the impact of each treatment on perioperative mortality and health care expenditures is unknown. The aim of the study was to compare mortality, morbidity and health care expenditures between LTA and PN. PATIENTS AND METHODS: A population-based assessment of 2471 patients with cT1a kidney cancer treated with either LTA or PN, between 2000 and 2009, in the SEER-Medicare database was performed. After propensity score matching, 30-day mortality, overall and specific complication rates, length of stay, readmission rates and health care expenditures according to LTA or PN were estimated. Multivariable logistic and linear models addressed the effect of each specific LTA approach on overall complication rates, length of stay, readmission rates and health care expenditures. RESULTS: The 30-day mortality was <2% after either LTA or PN (OR 2.27, p = 0.2). The overall complication rate was 21% after LTA and 40% after PN (OR 0.38, p < 0.001). Blood transfusions, infection/sepsis, wound infections, respiratory complications, gastrointestinal complications, acute kidney injury, and accidental puncture or laceration/foreign body left during procedure rates resulted lower after LTA relative to PN (all p < 0.05). Similarly, length of stay and health care expenditures resulted lower after LTA relative to PN (all p < 0.05). Conversely, readmission rate was not significantly different in LTA relative to PN (p = 0.1). CONCLUSIONS: Despite similar perioperative mortality, LTA is associated with lower complications rate, shorter length of stay and lower health care expenditure relative to PN.

Long-term survival of patients aged 80 years or older treated with radical prostatectomy for prostate cancer.

BACKGROUND: Radical prostatectomy (RP) is the gold standard for clinically localized prostate cancer (PCa) patients with life expectancy (LE) of at least 10 years. We examined long-term survival of men aged 80 years or older treated with RP and we attempted to identify criteria based on age and comorbidities that could predict survival of at least 10 years after RP, to identify those that might be considered for RP. PATIENTS AND METHODS: In Surveillance Epidemiology and End Results (SEER)-Medicare-linked database, we identified 234 octo- and nonagenarians with clinical T1, T2 or T3 PCa treated with RP between 1991 and 2009. Kaplan-Meier analyses examined 10-year survival patterns. Multivariable Cox regression analyses focused on the combined effect of age and/or Charlson Comorbidity Index (CCI) after adjusting for different confounders. RESULTS: The 10-year overall survival (OS) and cancer specific mortality (CSM) rates in the overall population were 51 and 9.9%. In individuals aged 80-81 years old, the 10-year OS was 62.4 vs. 39.6% in older patients (p = 0.001). Moreover, combination of age 80-81 with CCI = 0 yielded 10-year OS of 67.9 vs. 28.5% in older and sicker patients (p < 0.001). Age 80-81, absence of comorbidities and the combination of age 80-81 with CCI = 0, represented independent predictors of lower overall mortality (all p ≤ 0.01). CONCLUSIONS: Two out of three individuals selected for RP aged 80-81 years and without comorbidities, fulfill the criterion of LE of 10 years or more. Therefore, elderly PCa individuals can be suitable for surgical management, if appropriately selected, based on LE criterion.

Waterborne gemfibrozil challenges the hepatic antioxidant defense system and down-regulates peroxisome proliferator-activated receptor beta (PPARbeta) mRNA levels in male goldfish (Carassius auratus).

The lipid regulator gemfibrozil (GEM) is one of many human pharmaceuticals found in the aquatic environment. We previously demonstrated that GEM bioconcentrates in blood and reduces plasma testosterone levels in goldfish (Carassius auratus). In this study, we address the potential of an environmentally relevant waterborne concentration of GEM (1.5 microg/l) to induce oxidative stress in goldfish liver and whether this may be linked to GEM acting as a peroxisome proliferator (PP). We also investigate the autoregulation of the peroxisome proliferator-activated receptors (PPARs) as a potential index of exposure. The three PPAR subtypes (alpha, beta, and gamma) were amplified from goldfish liver cDNA. Goldfish exposed to a concentration higher (1500 microg/l) than environmentally relevant for 14 and 28 days significantly reduce hepatic PPARbeta mRNA levels (p<0.001). Levels of CYP1A1 mRNA were unchanged. GEM exposure significantly induced the antioxidant defense enzymes catalase (p<0.001), glutathione peroxidase (p<0.001) and glutathione-S-transferase (p=0.006) but not acyl-CoA oxidase or glutathione reductase. As GEM exposure failed to increase levels of thiobarbituric reactive substances (TBARS), we conclude that a sub-chronic exposure to GEM upregulates the antioxidant defense status of the goldfish as an adaptive response to this human pharmaceutical.

Stimulatory effect of the secretogranin-ll derived peptide secretoneurin on food intake and locomotion in female goldfish (Carassius auratus).

Secretoneurin (SN) is a conserved peptide derived by proteolytic processing from the middle domain of the ∼600 amino acid precursor secretogranin-II (SgII). Secretoneurin is widely distributed in secretory granules of endocrine cells and neurons and has important roles in reproduction as it stimulates luteinizing hormone release from the pituitary. A potential new role of SN in goldfish feeding is the subject of this study. Firstly, we established that acute (26 h; p<0.0001) and short-term (72 h; p=0.016) fasting increased SgIIa precursor mRNA levels 1.25-fold in the telencephalon, implicating SN in the control of feeding. Secondly, we determined that intracerebroventricular injections of the type A SN (SNa; 0.2 and 1 ng/g BW) increased food intake and locomotor behavior by 60 min. Fish injected with the lower and higher doses of SNa (0.2 and 1 ng/g) respectively exhibited significant 1.77- and 2.58-fold higher food intake (p<0.0001) than the saline-injected control fish. Locomotor behavior was increased by 1.35- and 2.26-fold for 0.2 ng/g SNa (p=0.0001) and 1 ng/g SNa (p<0.0001), respectively. Injection of 1 ng/g SNa increased mRNA levels of hypothalamic neuropeptide Y 1.36-fold (p=0.038) and decreased hypothalamic cocaine-and amphetamine-regulated transcript by 33% (p=0.01) at 2h and 5h post-injection, respectively. These data suggest interactions of SNa with stimulatory and inhibitory pathways of food intake control in fish.

Anxiolytic activity and active principles of Piper amalago (Piperaceae), a medicinal plant used by the Q'eqchi' Maya to treat susto, a culture-bound illness.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plant, Piper amalago L. (Piperaceae), is used traditionally by Q'eqchi' Maya healers for the treatment of "susto" a culture-bound syndrome. Previous research suggests that susto symptoms may be a manifestation of anxiety. The objectives were to characterize the effect of ethanolic extract of P. amalago in behavioral assays of anxiety at doses representative of traditional use and to isolate active principles. MATERIALS AND METHODS: Rats treated orally with low dose ethanolic extracts of P. amalago leaves (8-75mg/kg) were tested in several behavioral paradigms including the elevated plus maze (EPM), social interaction (SI), and conditioned emotional response (CER) tests, and compared to diazepam, a positive control. The active anxiolytic principle was isolated by bioassay guided isolation using an in vitro GABAA competitive binding assay. RESULTS: Extracts had significant anxiolytic activity in all behavioral tests, with the strongest activity in the SI and the CER paradigms. In an in vitro GABAA competitive binding assay, a 66.5µg/mL concentration of P. amalago ethanol extract displaced 50% of the GABAA-BZD receptor ligand [(3)H]-Flunitrazepam. Bioassay-guided fractionation identified a furofuran lignan, a molecule with structural similarity to yangambin, with high affinity for the GABAA-BZD receptor as the principle bioactive. CONCLUSION: The results suggest that the ethnobotanical use of this plant may have a pharmacological basis in its anxiolytic activity, as demonstrated in animal behaviour tests.

Stress elevates corticotropin-releasing factor (CRF) and CRF-binding protein mRNA levels in rainbow trout (Oncorhynchus mykiss).

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF)-binding protein (CRF-BP) cDNA and to examine the variations in CRF-BP and CRF mRNA levels in response to different intensities of stress. Trout were physically disturbed by a single or three consecutive periods of chasing until exhaustion followed by 2 h of recovery. The pituitary CRF-BP and preoptic area CRF1 mRNA contents were significantly increased only after repeated chasing events. Physical disturbance increased plasma cortisol levels with the largest change occurring in the group of trout that were exposed to repeated chasing events. Trout were also individually isolated in 120 l tanks or confined to 1.5 l boxes for 4, 24 or 72 h. CRF-BP mRNA levels in confined fish were greater than those of isolated fish at 72 h although there were no differences compared with the control group. CRF1 mRNA levels in the preoptic area were greater and remained elevated for a longer period in confined compared with isolated trout. Isolation led to a transient increase in plasma cortisol levels, but the higher cortisol values developed in the confined fish suggest that this treatment was more stressful than isolation. These results demonstrate that the intensity and duration of stress are important factors regulating CRF and CRF-BP mRNA levels in rainbow trout. We hypothesize that pituitary CRF-BP is involved in regulating the activity of the stress axis, possibly by reducing access to CRF1 receptors in the corticotropes.

Sex steroid regulation of brain glutamic acid decarboxylase (GAD) mRNA is season-dependent and sexually dimorphic in the goldfish Carassius auratus.

GABA, the major inhibitory neurotransmitter of the vertebrate brain, has been shown to play an important role in vertebrate reproduction by regulating LH release and sexual behavior. We have studied the expression of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in goldfish throughout the reproductive cycle in May (mature), November (early gonadal recrudescence) and February (late gonadal recrudescence) and in response to implanted sex steroids. Levels of GAD67 and GAD65 mRNA levels in the hypothalamus of both males and females were highest in the early stages of gonadal recrudescence. In the telencephalon, a different seasonal pattern of GAD expression was evident. The telencephalic expression GAD67, GAD65 and a novel isoform, GAD3, were highest in sexually mature fish in May. Five-day intraperitoneal implantation of gonad-intact fish with testosterone (T), estradiol (E2) or progesterone (P4) did not affect GAD expression in November and February. This is in contrast to results in May when sex differences in steroid responsiveness were evident. Progesterone decreased hypothalamic GAD67 and GAD65 in females and was without effect in males. All other treatments did not alter GAD67, GAD65 or GAD3 expression in the hypothalamus. Both T and P4 decreased GAD67 and GAD65 levels in the telencephalon of male goldfish but had no effect in females. Serum sex steroid levels in control and implanted mature males and females in May were similar so it is unlikely that sex differences in the GAD responses were a result of differences in serum sex steroid levels. These contrasting effects of sex steroids on males and females suggest important sex differences in the regulation of the GADs in sexually mature goldfish.

GABAergic modulation of the expression of genes involved in GABA synaptic transmission and stress in the hypothalamus and telencephalon of the female goldfish (Carassius auratus).

GABA is one of the most abundant neurotransmitters in the vertebrate central nervous system and is involved in neuroendocrine processes such as development, reproduction, feeding and stress. To examine the effect of GABA on gene expression in the brain, we used a cDNA macroarray containing 26 genes involved in GABA synaptic transmission (GABA receptor subunits, GABA transporters), reproduction (gonadotrophin-releasing hormone isoforms and oestrogen receptor alpha), feeding (neuropeptide Y and cholecystokinin), and stress [corticotrophin-releasing factor (CRF)]. To elevate GABA levels in the brain, we injected female goldfish with gamma-vinyl GABA (300 microg/g of body weight) (24 h), an irreversible inhibitor of the enzyme GABA transaminase (GABA-T). We found that increased levels of GABA in the hypothalamus resulted in a 2.2-fold down-regulation of GABA(A) receptor beta4 subunit mRNA. In the telencephalon, we found that increased GABA levels resulted in a 1.5-fold increase of CRF mRNA and a 1.8-fold decrease of GABA(A) receptor beta2 subunit mRNA. Increasing GABA in the hypothalamus and telencephalon of the goldfish did not significantly affect the mRNA abundance of genes involved in GABA synthesis (glutamic acid decarboxylase isoforms) and degradation (GABA-T), feeding, or reproduction. Our preliminary study suggests that the regulation of GABA receptor subunit mRNA expression by GABA may be a conserved evolutionary mechanism in vertebrates to modulate GABAergic synaptic transmission.

The human lipid regulator, gemfibrozil bioconcentrates and reduces testosterone in the goldfish, Carassius auratus.

Human and veterinarian pharmaceuticals have been detected in the aquatic environment for a number of years, but the potential for biological effects in exposed aquatic organism is only now being reported. The lipid regulator, gemfibrozil (GEM) is detected at microg/L concentrations in domestic wastewater and ng/L concentrations in surface waters. We investigated the uptake of GEM in goldfish (Carassius auratus) over a 96 h time period by measuring GEM in blood plasma using LC-MS/MS. Results indicated that GEM can be taken up from water through the gills. In goldfish exposed to GEM by a single intraperitoneal injection, concentrations of GEM in the blood plasma declined rapidly over 96 h post-injection, with a half-life estimated at approximately 19 h. Exposure of goldfish to waterborne GEM at an environmentally relevant concentration over 14 days resulted in a plasma bioconcentration factor of 113. In goldfish exposed to aqueous concentrations of GEM for 96 h or 14 days, plasma testosterone (T) was reduced by over 50% in fish from all treatments. As a possible mechanistic explanation for the observed reduction in T, levels of steroid acute regulatory (StAR) protein transcript in goldfish testes were assessed by RT-PCR. StAR protein is involved in the transport of cholesterol from the outer to the inner mitochondrial membrane for transformation by the first enzyme in steroidogenesis. After exposure to GEM for 96 h, a 50% decrease in StAR mRNA levels was observed in goldfish. Gonadal StAR mRNA levels were not affected in the 14 days exposure, indicating that the observed decreases in plasma testosterone were not solely due to impaired delivery of cholesterol to the inner mitochondrial membrane. Our results demonstrate that exposure to environmental levels of GEM leads to bioconcentration of the drug in plasma and the potential for endocrine disruption in fish.

Gamma-aminobutyric acid up-regulates the expression of a novel secretogranin-II messenger ribonucleic acid in the goldfish pituitary.

An RNA-arbitrarily primed PCR differential display strategy was used to identify candidate genes in the pituitary that are up-regulated by endogenously activated gamma-aminobutyric acid (GABA) systems that may also be involved in the control of reproduction. Goldfish were injected with the GABA metabolism inhibitor gamma-vinyl-GABA (GVG), known for its high efficiency to specifically increase endogenous brain and pituitary GABA levels in this species, resulting in higher levels of circulating gonadotropin-II (GTH-II). Several transcripts related to hormone secretion, signal transduction pathways, and messenger RNA (mRNA) editing were shown to be up-regulated after GVG injection. Among these transcripts we characterized an mRNA coding for the secretory vesicle protein secretogranin-II (SgII), a member of the chromogranin family, which is the precursor of a novel 34 amino acid neuropeptide, goldfish secretoneurin (SN). A semiquantitative PCR developed to measure pituitary SgII mRNA levels showed a 5-fold increase in GVG treated fish vs. control fish. Moreover, GVG treatment specifically increased SgII mRNA levels in gonadotrophs, concomitant with a decrease in GTH-II cell content. In addition, i.p. injection of synthetic goldfish SN increased GTH-II release in goldfish pretreated with the dopamine antagonist domperidone. Activation of GABAergic neurons has two effects, enhancing in vivo GTH-II release and up-regulating SgII mRNA specifically in goldfish gonadotrophs. Together with our SN bioactivity data, this suggests the existence in the pituitary of an autocrine or paracrine mechanism linked to the regulated secretory pathway in the gonadotrophs.