Laia Navarro-Martín

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles. II: Agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory conditions

Glyphosate-based herbicides are currently the most commonly used herbicides in the world. They have been shown to affect survival, growth, development and sexual differentiation of tadpoles under chronic laboratory exposures but this has not been investigated under more environmentally realistic conditions. The purpose of this study is (1) to determine if an agriculturally relevant exposure to Roundup WeatherMax®, a relatively new and understudied formulation, influences the development of wood frog tadpoles (Lithobates sylvaticus) through effects on the mRNA levels of genes involved in the control of metamorphosis; (2) to compare results to the well-studied Vision® formulation (containing the isopropylamine salt of glyphosate [IPA] and polyethoxylated tallowamine [POEA] surfactant) and to determine which ingredient(s) in the formulations are responsible for potential effects on development; and (3) to compare results to recent field studies that used a similar experimental design. In the present laboratory study, wood frog tadpoles were exposed to an agriculturally relevant application (i.e., two pulses) of Roundup WeatherMax® and Vision® herbicides as well as the active ingredient (IPA) and the POEA surfactant of Vision®. Survival, development, growth, sex ratios and mRNA levels of genes involved in tadpole metamorphosis were measured. Results show that Roundup WeatherMax® (2.89 mg acid equivalent (a.e.)/L) caused 100% mortality after the first pulse. Tadpoles treated with a lower concentration of Roundup WeatherMax® (0.21 mg a.e./L) as well as Vision® (2.89 mg a.e./L), IPA and POEA had an increased condition factor (based on length and weight measures in the tadpoles) relative to controls at Gosner stage (Gs) 36/38. At Gs42, tadpoles treated with IPA and POEA had a decreased condition factor. Also at Gs42, the effect on condition factor was dependent on the sex of tadpoles and significant treatment effects were only detected in males. In most cases, treatment reduced the normal mRNA increase of key genes controlling development in tadpoles between Gs37 and Gs42, such as genes encoding thyroid hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail. We conclude that glyphosate-based herbicides have the potential to alter mRNA profiles during metamorphosis. However, studies in natural systems have yet to replicate these negative effects, which highlight the need for more ecologically relevant studies for risk assessment.

Effects of the glyphosate-based herbicide Roundup WeatherMax ® on metamorphosis of wood frogs (Lithobates sylvaticus) in natural wetlands

Amphibian tadpoles develop in aquatic environments where they are susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are currently the most commonly used herbicide in the world and have been shown to affect survival and development of tadpoles under laboratory and mesocosm conditions. In the present study, whole wetland manipulations were used to determine if exposure to an agriculturally relevant application of Roundup WeatherMax(®), a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the development of wood frog tadpoles (Lithobates sylvaticus) under natural conditions. Wetlands were divided in half with an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at an environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and the predicted maximum environmental concentration (PMEC, 2.89 mg a.e./L), after which abundance, growth, development, and mRNA levels of genes involved in tadpole metamorphosis were measured. Results present little evidence that exposure to this herbicide affects abundance, growth and development of wood frog tadpoles. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research demonstrates that typical agricultural use of Roundup WeatherMax(®) poses minimal risk to larval amphibian development. However, our gene expression data (mRNA levels) suggests that glyphosate-based herbicides have the potential to alter hormonal pathways during tadpole development.

Efficient induction of spawning of Northern leopard frogs (Lithobates pipiens)during and outside the natural breeding season

BackgroundAmphibian declines are now recognized globally. It is also well known that many anurans do not reproduce easily in captivity, especially when held over long periods, or if they require hibernation before breeding. A simple method to induce spawning and subsequent development of large numbers of healthy tadpoles is therefore required to meet research and conservation goals.MethodsThe method is based on simultaneous injection of both female and male leopard frogs, Lithobates pipiens (formerly called Rana pipiens) with a cocktail of a gonadotropin-releasing hormone agonist (GnRH-A) and a dopamine antagonist. We call this the AMPHIPLEX method, which is derived from the combination of the words amphibian and amplexus. Following injection, the animals are thereby induced, and perform amplexus and natural fertilization under captive conditions.ResultsWe tested combinations of a GnRH agonist with 2 different dopamine antagonists in L. pipiens in the breeding season. The combination of des-Gly10, D-Ala6, Pro-NHEt9-GnRH (0.4 micrograms/g body weight; GnRH-A) with metoclopramide hydrochloride (10 micrograms/g body weight; MET) or domperidone (DOM) were equally effective, producing 89% and 88% successful spawning, respectively. This yielded more than 44,000 eggs for the 16/18 females that ovulated in the GnRH-A+MET group, and more than 39,000 eggs for the 15/17 females that ovulated in the GnRH-A+DOM group. We further tested the GnRH-A+MET in frogs collected in the wild in late autumn and hibernated for a short period under laboratory conditions, and report a low spawning success (43%). However, GnRH-A priming 24 hours prior to injections of the GnRH-A+MET cocktail in animals hibernated for 5–6 weeks produced out-of-season spawning (89%) and fertilization (85%) comparable to those we observed for in-season spawning. Assessment of age and weight at metamorphosis indicated that L. pipiens tadpoles resulting from out-of-season spawning grew normally and metamorphosed successfully.ConclusionWe provide evidence for successful captive breeding of the leopard frog, L. pipiens. This simple protocol can be used to obtain large numbers of eggs in a predictable, timed manner.

Sexing frogs by real-time PCR: using aromatase (cyp19) as an early ovarian differentiation marker.

Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, Lithobates sylvaticus and Silurana tropicalis tadpoles were sampled at different developmental stages covering the entire process of sex differentiation. Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase (cyp19), forkhead box L2 (foxl2) and the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17). Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of cyp17,cyp19 and foxl2 in L. sylvaticus adult gonads and gonad-mesonephros complex (GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S. tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future.

Early expression of aromatase and the membrane estrogen receptor GPER in neuromasts reveals a role for estrogens in the development of the frog lateral line system.

Estrogens and their receptors are present at very early stages of vertebrate embryogenesis before gonadal tissues are formed. However, the cellular source and the function of estrogens in embryogenesis remain major questions in developmental endocrinology. We demonstrate the presence of estrogen-synthesizing enzyme aromatase and G protein-coupled estrogen receptor (GPER) proteins throughout early embryogenesis in the model organism, Silurana tropicalis. We provide the first evidence of aromatase in the vertebrate lateral line. High levels of aromatase were detected in the mantle cells of neuromasts, the mechanosensory units of the lateral line, which persisted throughout the course of development (Nieuwkoop and Faber stages 34-47). We show that GPER is expressed in both the accessory and hair cells. Pharmacological activation of GPER with the agonist G-1 disrupted neuromast development and migration. Future study of this novel estrogen system in the amphibian lateral line may shed light on similar systems such as the mammalian inner ear.

Evidence for Alternative Splicing of a Dopamine D2 Receptor in a Teleost

Previous attempts at identifying an alternatively spliced dopamine (DA) D2 receptor in teleosts have proven unsuccessful. We provide evidence of a splicing event of a goldfish D2 (gfD2b1) receptor in the neuroendocrine brain of adult goldfish that produces a spliced short isoform (gfD2b1S). We also identify an additional novel D2b paralog (gfD2b2) that does not appear to be alternatively spliced in adult fish during the reproductive cycle. Relatively high mRNA levels of gfD2b1S were observed in the neuroendocrine brain and pituitary of sexually immature fish compared with sexually regressing fish. Real-time RT-PCR revealed that intraperitoneal injection of either SCH 23390 or sulpiride—D1- or D2-specific antagonists, respectively—decreased mRNA levels of gfD2b1S by 3.9-fold without affecting the unspliced isoforms. We suggest that the expression of the spliced D2 receptor modulates the inhibitory tone of DA throughout the reproductive cycle. The deduced amino acid sequence of gfD2b1S lacks 29 amino acids in the same region as the short isoform of mammalian D2. We propose that the gfD2b1S splice variant is the teleost ortholog of mammalian D2S. The hypothesis that D2 receptor splicing is a relatively recent innovation in higher tetrapods is not supported by our results.

Sodium perchlorate disrupts development and affects metamorphosis- and growth-related gene expression in tadpoles of the wood frog (Lithobates sylvaticus)

Numerous endocrine disrupting chemicals can affect the growth and development of amphibians. We investigated the effects of a targeted disruption of the endocrine axes modulating development and somatic growth. Wood frog (Lithobates sylvaticus) tadpoles were exposed for 2weeks (from developmental Gosner stage (Gs) 25 to Gs30) to sodium perchlorate (SP, thyroid inhibitor, 14mg/L), estradiol (E2, known to alter growth and development, 200nM) and a reduced feeding regime (RF, to affect growth and development in a chemically-independent manner). All treatments experienced developmental delay, and animals exposed to SP or subjected to RF respectively reached metamorphic climax (Gs42) approximately 11(±3) and 17(±3) days later than controls. At Gs42, only SP-treated animals showed increased weight and snout-vent length (P<0.05) relative to controls. Tadpoles treated with SP had 10-times higher levels of liver igf1 mRNA after 4days of exposure (Gs28) compared to controls. Tadpoles in the RF treatment expressed 6-times lower levels of liver igf1 mRNA and 2-times higher liver igf1r mRNA (P<0.05) at Gs30. Tadpoles treated with E2 exhibited similar developmental and growth patterns as controls, but had increased liver igf1 mRNA levels at Gs28, and tail igf1r at Gs42. Effects on tail trβ mRNA levels were detected in SP-treated tadpoles at Gs42, 40days post-exposure, suggesting that the chemical inhibition of thyroid hormone production early in development can have long-lasting effects. The growth effects observed in the SP-exposed animals suggest a relationship between TH-dependent development and somatic growth in L. sylvaticus tadpoles.

Characterization of multiple nestin isoforms in the goldfish brain.

Nestin is an intermediate filament protein involved in neurogenesis in fish, mice, and humans. In this study we used rapid amplification of cDNA ends PCR to isolate goldfish nestin (nes). PCR analysis and sequencing revealed three different nes transcripts of 4003, 2446, and 2126 nucleotides, which are predicted to generate proteins of 860, 274, and 344 amino acids in length. Sequence analysis suggests that these nes transcripts are likely a result of alternative splicing. We next applied a multiple-antigenic peptide strategy to generate a goldfish-specific nestin antibody. Western blotting with this antibody together with mass spectrometry verified the presence of major nestin protein isoforms with differing molecular weights (~70, 40 and 30kDa). We further examined expression patterns of these nestin protein isoforms in different parts of the goldfish brain and pituitary and found the telencephalon to express all three isoforms at a distinct level and abundance. We report that multiple nestin isoforms are present indicating another level of complexity for the regulation of intermediate filaments in comparison to mammals. Studying the differential roles and regulation of these nestins could lead to a better understanding of cellular remodeling during neurogenesis and the unparalleled regenerative abilities after damage in the teleost CNS.

Cloning, partial sequencing and expression analysis of the neural form of P450 aromatase (cyp19a1b) in the South America catfish Rhamdia quelen

Brain aromatase is a key enzyme exclusively expressed in fish radial glial cells that convert androgens into estrogens, thus controlling neuroendocrine functions and neurogenesis. As an important step in characterizing the neuroendocrine systems of Rhamdia quelen (jundiá), a partial cDNA sequence (1045 bp) of brain aromatase (cyp19a1b) was cloned and sequenced. At the nucleotide level the cDNA sequence was found to be 88% identical to cyp19a1b of two species of catfish, Ictalurus punctatus and Silurus meridionalis. The predicted amino acid sequence was between 80 and 91% similar to other teleosts. Real-time RT-qPCR analysis revealed that cyp19a1b was detected in pituitary, hypothalamus, telencephalon, head and posterior kidneys, liver and gonads (testis and ovary) of both males and females. The effects of E2 on cyp19a1b expression are sexually dimorphic in R. quelen. The injection of 17β-estradiol (E2) decreased head kidney mRNA levels of cyp19a1b in males and increased cyp19a1b mRNA levels in the pituitary and head kidney of females. This study demonstrated that the R. quelen cyp19a1b gene is expressed in brain, pituitary and peripheral tissues in both males and females.