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Team:Tuebingen/Project
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== '''Motivation''' == | == '''Motivation''' == | ||
| - | Why do want to establish a mechanism for steroid measurement? | + | Why do we want to establish a mechanism for steroid measurement? |
| - | Steroid hormones, especially estrogens, occur in all vertebrates | + | Steroid hormones, especially estrogens, occur in all vertebrates and play a crucial role in sexual differentiation. In recent times the pollution of waters with these hormones has become an increasing problem for the aquatic fauna. |
| - | Particularly waters adjacent to | + | Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations. |
| - | Scientific studies based on Danio rerio showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of Danio rerio leading to development of | + | Scientific studies based on ''Danio rerio'' showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of ''Danio rerio'' leading to development of ovotestis or complete feminization (Andersenc, 2002). |
| - | Intersex fish have | + | Intersex-fish have been reported in UK rivers since 1978 just downstream of an sewage treatment plant. |
| - | We believe that a first step | + | We believe that a first step in finding a solution to this environmental problem is an accurate and reliable method to quantify steroids which is easy to use and limited to an optical measurement. |
== '''Mechanism''' == | == '''Mechanism''' == | ||
Revision as of 17:41, 25 May 2012
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Overview
Our general aim is to establish a mechanism which allows us to define precisely a estrogen concentration in a dilute solution. Therefore we want to introduce the membrane progestin receptor (mPR) of the zebrafish (Danio rerio) in yeast (saccharomyces cerevisiae), linked to a reporter gene, which should allow us a quantitative measurement.
Motivation
Why do we want to establish a mechanism for steroid measurement?
Steroid hormones, especially estrogens, occur in all vertebrates and play a crucial role in sexual differentiation. In recent times the pollution of waters with these hormones has become an increasing problem for the aquatic fauna. Particularly waters functionalized by humans or adjacent to human settlements, for example in areas with agricultural use, show increased estrogen-concentrations.
Scientific studies based on Danio rerio showed that the consequences are devastating. High concentrations of 17α-ethinylestradiol, a hormone in most birth-control pills, affected the sex differentiation of Danio rerio leading to development of ovotestis or complete feminization (Andersenc, 2002). Intersex-fish have been reported in UK rivers since 1978 just downstream of an sewage treatment plant.
We believe that a first step in finding a solution to this environmental problem is an accurate and reliable method to quantify steroids which is easy to use and limited to an optical measurement.
Mechanism
Receptors
Membrangebunden, 7-Transmembranrezeptor (Innen C-Terminus, außen N-Terminus), PAQR-Familie (progesterone adiponectin Q receptor), Hly-III superfamily G-Protein gekoppelt: Aktivierung von inhibitorische Gi-Einheiten: Verringerung der Aktivität der Adenylyl-Cyclase G Proteins (Protein Data Bank) Artikel zu G-Proteinen - Struktur und Funktionsweise, schöne Bilder
Sequenz mPR alpha (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149121.1 NCBI],
Sequenz mPR beta (Zebrafisch): [http://www.ncbi.nlm.nih.gov/nuccore/AY149120.1 NCBI]
Homologe zum mPR Alpha Danio rerio:
- Goldfisch, Carassius auratus
- Katzenwels, Ictalurus punctatus
- Hundszungen, Cynoglossus semilaevis
- Flunder, Paralichthys lethostigma
- Meerforelle, Cynoscion nebulosus
- Buntbarsch, Oreochromis niloticus
- Umberfisch, Micropogonias undulatus
Zebrafrisch (Danio rerio) In Tübingen:
- Tübingen Map of the [http://wwwmap.tuebingen.mpg.de/ Zebrafish Genome]
- [http://www.mnf.uni-tuebingen.de/fachbereiche/biologie/institute/evolutionecology/lehrbereiche/physiologische-oekologie-der-tiere/staff/volker-scheil.html Dr. Volker Scheil], The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.
Inhibitor and promotor
Reporter gene
Measurement
References
Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment
- Diskussion: Wurde schon zuviel Geld, Mühe und Arbeit in die Erforschung von endokriner Disruption gesteckt?
- Die meisten Mensche erachten “hormonelle Verschmutzung” als geringfügiges Problem.
- Erste Beobachtung von intersexuellen Fischen (roach - Rotauge - Rutilis rutilis) 1978 in England stromabwärts einer Kläranlage.
- Vorkommen von intersexuellen Fischen abhängig vom Alter.
- Erste Studien zeigen einen kummulativen Effekt der endokrinen Substanzen.
- [http://de.wikipedia.org/wiki/Vitellogenine#Verwendung_als_Estrogen-Test Vitellogenin] als indikator für feminisierung/oestrogene. Nach zwei Wochen Abwasser -> 100.000-fache Konzentration in männlichen Fischen.
- Mitte der 90er: Intersexuelle Fische in ganz England verbreitet.
- Studien belegen den Verdacht auf den Einfluss durch [http://de.wikipedia.org/wiki/Estron Estron], [http://de.wikipedia.org/wiki/Estradiol Estradiol] (natürlich) und [http://de.wikipedia.org/wiki/Estradiol Ethinylestradiol] (synthetisch). Effekt im niedrigen ng/Liter bereich
- “male suppressing/female enhancing effect”
- Ebenfalls gemessene [http://de.wikipedia.org/wiki/Alkylophenol Alkylphenole], insbesondere [http://de.wikipedia.org/wiki/Nonylphenol Nonylphenol] und Oktylphenol haben östrogene Wirkung. Die benutzung dieser Stoffklasse ist in Europa bereits eingeschränkt, in den [http://water.epa.gov/scitech/swguidance/standards/criteria/aqlife/pollutants/nonylphenol/nonylphenol-fs.cfm USA] noch nicht.
- Östrogene beeinflussen die Expression Tausender von Genen
- Der Substanzmix im Gewässer erschwert die Detektion und Analyse: “it has proved extremely difficult to accurately determine the concentration of steroid oestrogens in effluents”
Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish (Polygenes Geschlechtsbestimmungssystem)
- Zebrafish sex is genetically determined with limited, secondary influences from the environment.
- No sign for CSD (chromosomal sex determination) was found in the species, which results in the conclusion, that Zebrafish might has a polygenic sex determination system.
- experiments were performed at Max-Planck-Institut für Entwicklungsbiologie in Tübingen, the zebrafish of different strains were bought from a local aquarium shop
- genomic DNA samples were extracted from tail fins
- “only” 89% of the DNA was restored
- there are no substantial differences between zebrafish male and female genomes and no dominant male/female genomes were discovered
- lack of oxygen level causes a reduction of estrogen synthesis → more androgen was built, which led to more male zebrafish in the population
Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
- Ergebnis der Studie: spezifische Aktivierung der Progestin Receptoren mPRa und mPRb durch Steroide (Progesteron, Cortisol, Testosteron, Estrogen, 11-Desoxycortisol) im Zebrafisch
-> MAPK (mitogen-activated ptotein kinase) Aktivierung bei gleichzeitigem Absenken der Adenylatcyclase Aktivität (über Pertussis Toxin-sensitiven Gi Pathway)
- Expression der der Rezeptoren in Säugetierbrustkrebszellen
- Überprüfung der Expression und Lokalisation der Rezeptoren mit
- Western blot
- Flow cytometry
- biotin surface labeling
Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen
- Hefe mit dem humanen Androgenrezeptor hAR (fest ins Genom integriert) und dem Reportergen LacZ (-> ß-Galactosidase)
- Rezeptor liegt allerdings nur im Cytoplasma vor, ist nicht membranstänig -> Problem, da Steroide in konjugierter Form nicht durch die ZM diffundieren können, aber im Urin sehr häuftig in dieser Form vorliegen
- -> weitere potentielle Anwendung für unser Projekt: Dopingtest für Anabolika? Wenn unsere Hefe mit unserem Rezeptor so funktioniert, wie wir uns es vorstellen, wäre das eine deutliche Verbesserung der in der Doktorarbeit beschriebenen Methode, Einsatz auch im humanen Leistungssport denkbar.
- -> evt. interessant für Sponsoren (Deutscher Olympischer Sport Bund, Nationale Anti Doping Agentur, ect.)
Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors
- menschliche mPRa, PRb, mPRg der Familie der Progestin und AdipoQ Rezeptoren (PAQR) wurden in Hefe exprimiert
- man wollte die genaue Rolle dieser Rezeptoren innerhalb des nicht genomischen Progesteron Signalweges herausfinden
- Bei Bindung von Liganden an die PAQR-Rezeptoren in Hefe wurde die FET3 Expression aktiviert
- -> mPRa, mPRb, mPRg hemmen allerdings die FET3 Expression
- -> die Expression von FET3 wird normalerweise durch den Osmotin Rezeptor in Hefe kontrolliert
- -> FET3 wurde somit als Reporter für die Funktionalität der PAQR-Rezeptoren verwendet
- “Assault on the male”, BBC 1996. Dokumentation zum Thema http://www.youtube.com/watch?v=LkxIJJI37bQ
- Dr. Volker Scheil, The impact of potential environmental stressors on early development and cellular and biochemical biomarkers in fish, Main research: Fish embryotoxicity, histopathology and stress protein (hsp 70) responses.
- Harris et al. - 2011 - The consequences of feminization in breeding groups of wild fish
- Liew et al. - 2012 - Polygenic Sex Determination System in Zebrafish
- Sumpter, Johnson - 2008 - 10th Anniversary Perspective Reflections on endocrine disruption in the aquatic environment from known knowns to unknown unknowns (and many things in between)
- Werner, Palace, Wautier - 2006 - Reproductive fitness of lake trout (Salvelinus namaycush) exposed to environmentally relevant concentrations of the potent estrogen ethynylestradiol (EE2) in a whole lake exposure experiment
- Fenske, M., Maack, G., Schäfers, C., & Segner, H. (2005). An environmentally relevant concentration of estrogen induces arrest of male gonad development in zebrafish, Danio rerio. Environmental toxicology and chemistry / SETAC, 24(5), 1088-98. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/16110986 (kein PDF)
- Schäfers, C., Teigeler, M., Wenzel, A., Maack, G., Fenske, M., & Segner, H. (2007). Concentration- and time-dependent effects of the synthetic estrogen, 17alpha-ethinylestradiol, on reproductive capabilities of the zebrafish, Danio rerio. Journal of toxicology and environmental health. Part A, 70(9), 768-79. doi:10.1080/15287390701236470
- Segner, H., Caroll, K., Fenske, M., Janssen, C. R., Maack, G., Pascoe, D., Schäfers, C., et al. (2003). Identification of endocrine-disrupting effects in aquatic vertebrates and invertebrates: report from the European IDEA project. Ecotoxicology and environmental safety, 54(3), 302-14. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12651186 (kein PDF)
- Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
- Thomas et al. - 2007 - Steroid and G protein binding characteristics of the seatrout and human progestin membrane receptor alpha subtypes and their evolutionary origins
- Thomas - 2008 - Characteristics of membrane progestin receptor alpha (mPR ) and progesterone membrane receptor component 1 (PGMRC1) and their roles in mediating rapid progestin actions
- Jessica L. Smith, Brian R. Kupchak, Ibon Garitaonandia, L. Kim Hoang, Andrew S. Maina, Lisa M. Regalla, and Thomas J. *Lyons - 2008 - Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors
