Developing new model organisms
National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3R's)
Principal investigator: Susan Jobling
Co-investigator: Dr Edwin Routledge
Declining male reproductive health is of particular current concern, underlining the need for continued focus on the development of pharmaceuticals with which to treat male reproductive disorders and diseases. Moreover, scientific research on the relationship between the environment and health has revealed that a larger proportion of these disorders may be caused by exposure to environmental pollutants than was previously thought. This has led to changes in legislation concerning the safety testing of chemicals, which invariably involves increased numbers of vertebrate animal being used in chemical and pharmaceutical testing.
In keeping with strategies to replace, reduce and refine in animal work, we investigated the potential use of gastropod molluscs as alternative models for male reproductive toxicity testing, due to some similarity between their reproductive anatomy with that of mammals, together with recently published evidence that endocrine disrupting chemicals can cause similar biological effects in gastropods to those seen in mammals.
To test this hypothesis, we used the freshwater pulmonate snail, Biomphalaria glabrata, for which various genetic tools and a draft genome had recently become available, to investigate the effects of two steroid androgens on the development of secondary sexual organs and gonads. Gastropod snails were exposed to two different steroid androgens 1) a synthetic anabolic steroid: 17α-methyltestosterone and 2) the natural potent vertebrate steroid: 5α-dihydrotestosterone. In vertebrates developmental stages and puberty are considered the most sensitive period likely to be affected by endocrine disrupting chemicals. Therefore the snails were continuously exposed to each androgen throughout embryonic development and up to sexual maturity. Exposure concentrations were chosen to reflect doses previously published to elicit reproductive effects in other gastropod species and in fish. Morphological (reproductive organ growth), histopathological (cellular level) and differential gene expression analysis revealed no significant differences between exposed and control tissues from snails exposed to 17α-methyltestosterone and 5α-dihydrotestosterone, suggesting that the snails did not respond to, and were not affected by, exposure to these androgens.
In parallel to the experimental work, we also used a molecular bioinformatic approach to investigate if gastropod molluscs such as B. glabrata contained the androgenic pathways necessary to replace vertebrate test systems. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated. Both the L. gigantean genome and the B. glabrata draft genome were exhaustively searched for nuclear receptors and we discovered that although they possess homologues to many vertebrate and other invertebrate nuclear receptors, neither gastropod possessed an androgen receptor, via which steroid androgens elicit androgenic effects (such as male reproductive growth) in vertebrates. This was an important revelation and substantiates our negative effect of androgens finding from our experimental studies.
Both the experimental and bioinformatics findings are extremely interesting as it has been a long held view of some researchers that gastropod molluscs use vertebrate type androgens in reproduction; and therefore would be reproductively stimulated or disrupted by classic androgens or anti-androgens. Our work highlights the importance of having mechanistically relevant endpoints in toxicological testing strategies.
Biological Pathways and Systems – Same but different?
In total thirty-nine and thirty-three putative nuclear receptors were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major nuclear receptor subfamilies (1-6). Many of the identified nuclear receptors are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets (e.g. Androgen, Progesterone, Glucocorticoid receptors). The mollusc genomes also contain nuclear receptor homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96).
The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different to mammals.