Team:Tuebingen/Project

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Overview

Our general aim is to establish a simple mechanism which allows us to measure an estrogen concentration in a solution as precisely as possible. Therefore we want to clone the membrane progestin receptor (mPR) of the zebrafish (Danio rerio) in yeast (saccharomyces cerevisiae) and link it to a reporter gene, which allows 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 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.

Occurring Questions

On our way designing the major pathway to express a specific reporter gene to demonstrate the presence of steroid hormones, we had and still have to deal with several questions concering the choice of BioBricks, genes and vectors to construct a firm method to determine "pollution" by steroid hormones. As a conclusion, we have to meet two major requirements for our system:

It should be as cost-efficient as possible for easy and regular application
It should be resistant to yeast's own metabolism. (Not be disturbed by unexpected occuring expression)

At first we had to find an appropriate receptor to detect steroid hormones.

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): NCBI,

Sequenz mPR beta (Zebrafisch): 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 Zebrafish Genome
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
Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish
Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen
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
“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

@@ Line 58: / Line 58: @@
 == '''References''' ==
-=== Sumpter, Johnson (2008) Reflections on endocrine disruption in the aquatic environment ===
+*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?
+*Liew et. al. - 2012 Polygenic Sex Determination System in Zebrafish
-*Die meisten Mensche erachten “hormonelle Verschmutzung” als geringfügiges Problem.
+*Hanna et al. - 2006 - Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors alpha and beta in transfected cells
-*Erste Beobachtung von intersexuellen Fischen (roach - Rotauge - Rutilis rutilis) 1978 in England stromabwärts einer Kläranlage.
+*Reupke - 2011 - Detektion dopingrelevanter anaboler Steroide in Pferdeurin und Pferdeplasma mithilfe eines Reportergen-Assays in Hefezellen
-*Vorkommen von intersexuellen Fischen abhängig vom Alter.
+*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
-*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.