Laser capture microdissection of gonads from juvenile zebrafish

<p>Abstract</p> <p>Background</p> <p>Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex determination and differentiation.</p> <p>Methods</p> <p>The laser capture microdissection technique enables isolation of specific cells and tissues and thereby removes the noise of gene expression from other cells or tissues in the gene expression profile. A protocol developed for laser microdissection of human gonocytes was adjusted and optimised to isolate juvenile zebrafish gonads.</p> <p>Results</p> <p>The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows staining, identification, isolation and subsequent RNA purification and amplification of gonads from individual juvenile zebrafish thereby enabling gonadal gene expression profiling.</p> <p>Conclusion</p> <p>The study presents a protocol for isolation of individual juvenile zebrafish gonads, which will enable future investigations of gonadal gene expression during the critical period of sex differentiation. Furthermore, the presented staining method is applicable to other species as it is directed towards alkaline phosphatase that is expressed in gonocytes and embryonic stem cells, which is conserved among vertebrate species.</p

Expression profiles for six zebrafish genes during gonadal sex differentiation

<p>Abstract</p> <p>Background</p> <p>The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation.</p> <p>Results</p> <p>In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish.</p> <p>Conclusion</p> <p>In zebrafish, the first significant peak in gene expression during the investigated period (2–40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex differentiation of males.</p

Values are expressed as arbitrary units of expression levels normalised against the expression levels of amplified from the same template

* indicate significant difference (p < 0.05) after Tukey test compared to the day before in the high expresser group. There was always significant difference between the high and low expresser group (p < 0.05) after Tukey test.<p><b>Copyright information:</b></p><p>Taken from "Expression profiles for six zebrafish genes during gonadal sex differentiation"</p><p>http://www.rbej.com/content/6/1/25</p><p>Reproductive biology and endocrinology : RB&E 2008;6():25-25.</p><p>Published online 30 Jun 2008</p><p>PMCID:PMC2500022.</p><p></p

Development of the integrated fish endocrine disruptor test (iFEDT)-Part A:Merging of existing fish test guidelines

There has been increasing interest in endocrine-disrupting chemicals (EDCs) among scientists and public authorities over the last 30 years, notably because of their wide use and the increasing evidence of detrimental effects on humans and the environment. However, test systems for the detection of potential EDCs as well as testing strategies still require optimization. Thus, the aim of the present project was the development of an integrated test protocol that merges the existing OECD test guidelines (TGs) 229 (fish short-term reproduction assay) and 234 (fish sexual development test) and implements thyroid-related endpoints for fish. The integrated fish endocrine disruptor test (iFEDT) represents a comprehensive approach for fish testing, which covers reproduction, early development, and sexual differentiation, and will thus allow the identification of multiple endocrine-disruptive effects in fish. Using zebrafish (Danio rerio) as a model organism, two exposure tests were performed with well-studied EDCs: 6-propyl-2-thiouracil (PTU), an inhibitor of thyroid hormone synthesis, and 17α-ethinylestradiol (EE2), an estrogen receptor agonist. In part A of this article, the effects of PTU and EE2 on established endpoints of the two existing TGs are reported, whereas part B focuses on the novel thyroid-related endpoints. Results of part A document that, as expected, both PTU and EE2 had strong effects on various endocrine-related endpoints in zebrafish and their offspring. Merging of TGs 229 and 234 proved feasible, and all established biomarkers and endpoints were responsive as expected, including reproductive and morphometric changes (PTU and EE2), vitellogenin levels, sex ratio, gonad maturation, and histopathology (only for EE2) of different life stages. A validation of the iFEDT with other well-known EDCs will allow verification of the sensitivity and usability and confirm its capacity to improve the existing testing strategy for EDCs in fish. Integr Environ Assess Manag 2023;00:1-13. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology &amp; Chemistry (SETAC).</p

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays

New approach methods to improve human health risk assessment of thyroid hormone system disruption–a PARC project

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available–and are currently in the process of regulatory validation–there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays