Elsevier

Aquatic Toxicology

Volume 86, Issue 2, 31 January 2008, Pages 131-141
Aquatic Toxicology

Development of a marine fish model for studying in vivo molecular responses in ecotoxicology

https://doi.org/10.1016/j.aquatox.2007.10.011Get rights and content

Abstract

A protocol for fixation and processing of whole adult marine medaka (Oryzias melastigma) was developed in parallel with in situ hybridization (ISH) and immunohistochemistry (IHC) for molecular analysis of in vivo gene and protein responses in fish. Over 200 serial sagittal sections (5 μm) can be produced from a single adult medaka to facilitate simultaneous localization and quantification of gene-specific mRNAs and proteins in different tissues and subcellular compartments of a single fish. Stereological analysis (as measured by volume density, Vv) was used to quantify ISH and IHC signals on tissue sections. Using the telomerase reverse transcriptase (omTERT) gene, omTERT and proliferating cell nuclear antigen (PCNA) proteins as examples, we demonstrated that it is possible to localize, quantify and correlate their tissue expression profiles in a whole fish system. Using chronic hypoxia (1.8 ± 0.2 mg O2 L−1 for 3 months) as an environmental stressor, we were able to identify significant alterations in levels of omTERT mRNA, omTERT protein, PCNA (cell proliferation marker) and TUNEL (apoptosis) in livers of hypoxic O. melastigma (p < 0.05). Overall, the results suggest that O. melastigma can serve as a model marine fish for assessing multiple in vivo molecular responses to stresses in the marine environment.

Introduction

A variety of molecular, biochemical and histological responses in fish have been employed as biomarkers of various environmental stresses (Gavilán et al., 2001, Au, 2004, Facey et al., 2005, Hutchinson et al., 2006). While these sub-organismal responses are sensitive, reproducible and easier to determine, most of them are tissue- and/or cell-specific. For instance, CYP1A1 mRNA expression and EROD enzyme induction are different in liver (hepatocytes), intestine (enterocytes) and gill (epithelia) of fish, depending on the exposure route of toxicants (Wong et al., 2001, Yuen and Au, 2006). Current molecular techniques such as real-time PCR and Western blotting and chemical analysis of metabolites normally only provide information for individual tissues or the whole animal, but are unable to differentiate molecular changes among different cell types within the tissue.

Recently, there is a trend of using small size fish as sentinel vertebrate species for ecotoxicology and biomedical research (Moore, 2002, Wittbrodt et al., 2002, Hawkins et al., 2003, Hinton et al., 2005). Small fish have several advantages in ecotoxicological studies, since they are generally easy to maintain and breed under laboratory conditions. The generation time is relatively short, and fish can produce eggs regularly, hence providing a variety of developmental and reproductive endpoints for whole life cycle and multi-generation assessments. To this end, the zebrafish (Danio rerio), fathead minnow (Pimephales promelas), mosquito fish (Gambusia affinis), guppy (Poecilia reticulata) and Japanese medaka (Oryzias latipes) have been commonly used as freshwater fish models in ecotoxicological studies (Dodd et al., 2000, Castro et al., 2004, Wolf et al., 2004, Volz et al., 2005, Carter and Wilson, 2006, Kissling et al., 2006). Surprisingly, a fish model for assessing environmental stress in the marine environment has not been developed, although a number of freshwater/estuarine species (e.g. the sheepshead minnow Cyprinodon variegatus and the mummichog Fundulus heteroclitus) has also been commonly adapted to seawater for toxicological studies due to their hardiness in captivity. However, both C. variegatus and F. heteroclitus suffer from varying rates of growth, which compromises equilibrating toxic responses, and poor knowledge of their genomes make molecular toxicological studies on these species difficult. Further, the mummichog takes 2 years to reach sexual maturity, and this makes it difficult to carry out whole life cycle studies.

The marine medaka Oryzias melastigma (McClelland) has a number of attributes that render it a potentially good marine fish for ecotoxicological studies. O. melastigma is small and easy to culture and breed, and it completes the whole life cycle in seawater. Marine medaka are similar to freshwater medaka O. latipes, they exhibit uniform growth, which confer an additional advantage in using this species for ecotoxicological studies. Phylogenetically, this species is closely related to O. latipes, of which the entire genome has been worked out recently (Kasahara et al., 2007). The anatomy, biology and nutritional requirements of O. melastigma is similar to that of O. latipes, which are well known and an atlas is available (Anken and Bourrat, 1998, Fujita et al., 2006). In addition, much of the information on the physiology of O. latipes (http://www.bio.nagoya-u.ac.jp:8000/Welcome.html) is also applicable for O. melastigma.

Notwithstanding, the use of small fishes for tissue-specific molecular analyses presents a major challenge. The quantity of a specific tissue available for analysis is often very limited, and isolation of such a small amount of tissue is often difficult and time consuming. However, this limitation can be overcome by using in situ hybridization (ISH) and immunohistochemistry (IHC) analyses on preserved whole fish tissues. With the recent advent of image analysis software, the effectiveness of stereological analysis for quantification of IHC and ISH signals on tissue sections has been greatly enhanced. Moreover, various technical problems are associated with the fixation and sectioning of relatively large-sized adult fish specimens (ca. 3 mm) (Weber et al., 2002), due mainly to its heavy bony structures. Traditional decalcification of bony structures using formic acid or EDTA not only lead to poor RNA preservation, but are also time consuming (and may require up to 7 days) (Moore et al., 2002). Previous fixation protocols for adult small fish, e.g. the Japanese medaka, zebrafish, guppy and mosquito fish were specifically designed for histopathologic evaluation (Fournie et al., 1996, Neely et al., 2002, Zodrow et al., 2004) and a few on immuno-localization studies (Ortego et al., 1994, Moore et al., 2002, Ko et al., 2005). Until now, no protocols have been developed for the parallel detection of mRNA and protein molecules in whole tissues of these adult small fish. In the first part of this study, we have developed and optimized procedures for the fixation and processing of whole adult marine medaka, enabling the production of tissue sections suitable both for subsequent ISH and IHC analyses.

Telomerase is an enzyme involved in cell immortalization, carcinogenesis and tissue regeneration (Greider, 1998). The catalytic subunit telomerase reverse transcriptase (TERT) has been shown to regulate cell proliferation, mediate apoptosis, promote DNA repair and cell survival in vitro (see review of Chung et al., 2005), which suggest that TERT has a central role in controlling in vivo cell growth and tissue homeostasis. In fish, expression of TERT gene has been found in a variety of somatic tissues in O. melastigma (Yu et al., 2006) and Fugu rubripes (Yap et al., 2005). In the second part of this study, we employed the omTERT mRNA and protein and proliferating cell nuclear antigen (marker for cell proliferation) as molecular endpoints to demonstrate the feasibility of using ISH and IHC techniques to simultaneously localize and measure in vivo expression levels of these gene and proteins in different tissues of a single medaka fish, and to allow statistical analysis to be made on the correlation of these gene and protein expression data in a whole fish.

Hypoxia has now become a pressing environmental problem in aquatic systems worldwide (Diaz, 2001, Wu, 2002). Hypoxia has been reported to up-regulate TERT expression in liver of O. melastigma (through the hypoxia-inducible factor-1, HIF-1) (Yu et al., 2006), which may perturb normal cell proliferation and apoptosis in hepatocytes. In the third part of this report, we used hypoxia as a model stressor and the liver as a model organ to study the stress responses of omTERT mRNA (by ISH) and protein (by IHC), cell proliferation (by PCNA) and apoptosis (by the terminal dideoxynucleotidyl-mediated dUTP nick end labeling, TUNEL) in O. melastigma. The overall objective of this study is to demonstrate that O. melastigma can serve as a good marine fish model for ecotoxicology, and enable in vivo molecular responses at the nucleic acid and protein levels to be localized and measured simultaneously in different tissues/cells of the same individual.

Section snippets

Marine medaka

Marine medaka were purchased from a commercial hatchery in Taiwan and were maintained in 30‰ artificial seawater at 5.8 ± 0.2 mg O2 L−1, 28 ± 2 °C in a 14-h light:10-h dark cycle. Under optimal growth and breeding conditions, adult O. melastigma produced sufficient numbers of genetically homogeneous offspring. For the chronic hypoxia exposure experiment, 4-week old O. melastigma were used and divided into two groups. The first group was maintained in a hypoxic system (1.8 ± 0.2 mg O2 L−1) and the second

Whole adult O. melastigma fixation and processing

The protocol that yielded high quality tissue sections with optimal preservation of tissue and cellular morphology, nucleic acid and antigenicity was developed by optimizing various fixation and processing regimes that included testing the efficacy of: (i) fixative combinations; (ii) clearing media and (iii) processing temperatures (Table 1). Among all of the conditions tested, primary fixation of adult O. melastigma in a cocktail of 0.05% glutaraldehyde (Glut), 2% paraformaldehyde (PFA) and 1%

Discussion

The fixation and processing protocol developed for adult whole O. melastigma in this study is reproducible and cost-effective for ISH and IHC analyses of the omTERT gene and protein, PCNA and TUNEL assay in multiple organs of a single fish. Applying these techniques to O. melastigma, we have demonstrated the feasibility of simultaneous expression and quantification analyses of multiple genes/proteins in whole fish sections without needing to dissect out individual organs for tissue processing,

Acknowledgements

This study was supported by a grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project no. AoE/P-04/04) and CityU grants (Project nos. 7001834 and 7002117). The research was also supported, in part, by a grant from the US EPA STAR grant no. R-831846. The authors would like to thank Prof. Juro Koyama, Kagoshime University, Japan, for introducing us the O. melastigma.

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