In Vivo Screening Of Environmental Estrogens Using Chromadorina Germanica As Model Animal

Currently, the most mature established rapid screening method to detect potential environmental estrogens can be divided into two categories, which are in vitro screening and in vivo screening. In vitro screening is quicker and easier; while in vivo screening reflects the effects and mechanisms on the whole organism of environmental estrogens better, which is generally considered to be more reliable. Therefore, screening methods employing whole animal in vivo models is of more importance and practical significance. However, rodent and fish, which are widely used for in vivo screening, have disadvantages such as long experimental periods and high costs in application. Free-living marine nematode can be chosen as a model organism to solve this problem. Free-living marine nematode is an essential constituent of Nematode, which distributes extensively, range from coastal intertidal tide line to the deepest deep ocean trench, and from the cold poles to the deep-sea ridges of high-temperature. Free-living marine nematode Chromadorina germanica is small, gonochorism with bisexual reproduction, and has a short life cycle (14d), which has been single-specie continuous cultured for more than 90 generations in our laboratory.Vitellogenin (VTG) is an important biomarker of environmental estrogens screening, especially male fish VTG mRNA, which has high sensitivity and specificity. Environmental estrogens screening methods, with VTG mRNA as a biomarker, include real-time quantitative PCR, Northern blot, in situ hybridization. In situ hybridization is a technology which used to detect target nucleic acid with complementary labeled nucleic acid probe in cells or tissues. This technique not only conveniently exempts from extracting RNA from tissues, but also provide a accurate and directviewing reflection of the location and status of VTG mRNA in biological samples.Monocrotophos is a highly effective organophosphorus pesticide, of which residues in environment have threated the health of aquatic organisms or even human. Bing Xin's study showed that monocrotophos induced synthesis and secretion of vitellogenin in male goldfish, and turned out to be an environmental estrogen. Therefore, this thesis has been designed to detect induced-expression of VTG mRNA in C. germanica exposured to endogenous estrogen (E2) and environmental estrogen (monocrotophos), by using in situ hybridization. The feasibility of constructing a novel in vivo rapid screening method of detecting environmental estrogens with C. germanica as model animal and VTG mRNA as molecular biomarker.The main outcomes are as follows:Degenerate primers for cloning C. germanica VTG gene has been designed, and a 525bp long nucleotide sequence has been obtained by PCR, RT-PCR and so on. Comparison results between this nucleic acid sequence and VTG gene sequences of other nematodas on NBCI website and in biological analysis software DNAman show that this nucleic acid fragment is a part of C. germanica vit-6 sequence.According to the available sequence of C. germanica vit-6 gained above, C. germanica VTG mRNA oligonucleotide probes labeled with DIG have been designed and prepared. Application of these probes in in situ hybridization experiments demonstrates that there is specificity hybridization between the DIG-labeled probe and VTG mRNA in C. germanica.Based on the DIG-labeled oligonucleotide probes, more sensitive probes labeled directly with fluorescein 5-FAM has been synthesised and applied in fluorescence in situ hybridization. The results of hybridization not only have further verified the specificity between the oligonucleotide probes and C. germanica VTG mRNA, but also shows a slight upward trend of synthesis of VTG mRNA in male C. germanica with the increase of the concentration of environmental estrogens.In summary, in situ hybridizations using specificity probes which have been designed based on cloned C. germanica VTG gene fragment, have effectively detected the induced-expression of VTG mRNA by environmental estrogen in male C. germanica. Using free-living marine nematodes C. germanica as model animal to construct a novel in vivo rapid screening model of detecting environmental estrogens is practicable and of application value.