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Since we want to measure progestin concentrations (e.g. Kolodziej et al., 2003, also see Motivation) that have an effect on fish but usually do not harm humans (European Agency for the Evaluation of Medicinal Products, 1999) we decided to not use human progestin receptors (for a human cell derived measurement system see for example van der Linden et al., 2008) but rather the progestin receptors of fish for our progestin measurement system. This has some advantages:

Firstly, any progestin concentration that can cause a reporter signal in our system might also affect fish in some way. In fact, we use exactly the progestin receptors that induce the oocyte meiotic maturation in fish and amphibia we mentioned in our project motivation (Zhu et al., 2003).

Secondly, currently unknown compound that can bind to the progestin receptor may have progestin-like effects in fish (see the relation between bisphenol A and estrogen receptors (Takayanagi et al., 2006, Gould et al., 1998, Okada et al., 2008) as an example for such reactions).

We are using progesterone membrane receptors (mPR) that mediate nongenomic progesterone responses and belong to the progestin and AdipoQ-Receptor (PAQR) family (Tang et al., 2005) as “progestin sniffers” for our measurement system. mPR seems to have G-protein coupled receptor (GPCR) characteristics though there are no other GPCRs in the PAQR family (Tang et al., 2005, Zhu et al., 2003).

Smith et al. (2008) were able to show that human mPRs can sense progesterone and structurally similar compounds like 17alpha-Hydroxyprogesterone at physiologically relevant concentrations, therefore we believe that other compounds that are related to progesterone can bind to mPRs as well. In their study on female Xenopus laevis Liu and Patiño (1993) found such a correlation between structural similarity and binding affinity.

We chose Saccharomyces cerevisiae as chassis for our biological system due to the fact that yeast possesses endogenous receptors of the PAQR family (Lyons et al., 2004) thus the apparatus for the operation of receptors of that family should be available in these organisms. In fact, Kupchak et al. (2007) showed that many PAQR proteins are able to “activate similar intracellular signaling cascades, suggesting a conserved mechanism for signal transduction”. Also, S. cerevisiae does not use progesterone in its own metabolism and does not possess any progesterone receptors thus should not interfere with our measurement system (Smith et al., 2008). Furthermore S. cerevisiae is a rather well understood eukaryote organism and offers acceptable reproduction rates. We did not consider Escherichia coli since “it is not known if PAQR receptors function the same in this organism as they do in eukaryotes” (Smith et al., 2008). All in all, S. cerevisiae is an ideal chassis organism for our measurement system.

Kupchak et al. (2007) showed that yeast PAQR proteins repress a gene called FET3 in S. cerevisiae. Knowing this, Smith et al. (2008) used the expression of FET3 as a reporter for their study of human mPRs (which were heterologically expressed in yeast) and were able to show that these mPRs, too, repress FET3. Since both yeast and human PAQR proteins reliably repress FET3 we are confident that PAQR proteins from other species will interact with the FET3 promoter, too. In fact, Zhu et al. (2003) obtained evidence that teleost fishes (like Danio rerio) employ mPRs for the induction of oocyte meiotic maturation. Amphibians (like Xenopus laevis) use high-affinity mPRs for the induction of the same process (Liu and Patiño, 1993).

For our measurement system we decided to use membrane progestin receptors (mPR) of Xenopus laevis and Danio rerio as progestin detectors / “sniffers”. In the end when our system is ready for use we will analyze the properties of both mPRs (denoted mPR Xl and mPR Dr) and decide which one suits our needs best. Once progestin binds to the mPR the FET3 gene promoter (Pfet3) will be repressed by an endogenous signaling molecule from the PAQR-pathway of our chassis (S. cerevisiae). As a result the expression of downstream genes of Pfet3 will stop. In order to receive a positive feedback when progestin is detected we need to invert the receptor signal – therefore we have combined Pfet3 with a repressor that represses the promoter of our reporter.

The mPRs themselves are regulated by the constitutive promoter of the ADH1 gene (Padh1), which we created by PCR of yeast DNA. The DNA-sequence of our Padh1 is identical to BBa_J63005.

Additional information:
The FET3 protein is an essential element of S. cerevisiae’s iron metabolism. FET3 is a multicopper oxidase that is responsible for high-affinity ferrous iron (Fe(II)) uptake in yeast (Askwith et al., 1994). FET3 is metalloregulated which means that FET3 is expressed when intracellular metal levels (e.g. zinc, iron) are low but repressed when there is an abundance of metals (Lyons et al., 2004). Since iron is a main cause for FET3 repression all measurements with our system must be conducted in low iron medium (LIM).

References

ASKWITH, C., EIDE, D., VAN HO, A., BERNARD, P. S., LI, L., DAVIS-KAPLAN, S., SIPE, D. M. & KAPLAN, J. 1994. The FET3 gene of S. cerevisiae encodes a multicopper oxidase required for ferrous iron uptake. Cell, 76, 403-410.

EUROPEAN AGENCY FOR THE EVALUATION OF MEDICINAL PRODUCTS. 1999. Progesterone - Summary Report [Online]. London. Available: http://www.ema.europa.eu/docs/en_GB/document_library/Maximum_Residue_Limits_-_Report/2011/07/WC500108427.pdf.

GOULD, J. C., LEONARD, L. S., MANESS, S. C., WAGNER, B. L., CONNER, K., ZACHAREWSKI, T., SAFE, S., MCDONNELL, D. P. & GAIDO, K. W. 1998. Bisphenol A interacts with the estrogen receptor α in a distinct manner from estradiol. Molecular and Cellular Endocrinology, 142, 203-214.

KOLODZIEJ, E. P., GRAY, J. L. & SEDLAK, D. L. 2003. Quantification of steroid hormones with pheromonal properties in municipal wastewater effluent. Environmental Toxicology and Chemistry, 22, 2622-2629.

KUPCHAK, B. R., GARITAONANDIA, I., VILLA, N. Y., MULLEN, M. B., WEAVER, M. G., REGALLA, L. M., KENDALL, E. A. & LYONS, T. J. 2007. Probing the mechanism of FET3 repression by Izh2p overexpression. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1773, 1124-1132.

LIU, Z. & PATIÑO, R. 1993. High-affinity binding of progesterone to the plasma membrane of Xenopus oocytes: characteristics of binding and hormonal and developmental control. Biology of Reproduction, 49, 980-988.

LYONS, T. J., VILLA, N. Y., REGALLA, L. M., KUPCHAK, B. R., VAGSTAD, A. & EIDE, D. J. 2004. Metalloregulation of yeast membrane steroid receptor homologs. Proceedings of the National Academy of Sciences of the United States of America, 101, 5506-5511.

OKADA, H., TOKUNAGA, T., LIU, X., TAKAYANAGI, S., MATSUSHIMA, A. & SHIMOHIGASHI, Y. 2008. Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma. Environ Health Perspect, 116, 32-8.

SMITH, J. L., KUPCHAK, B. R., GARITAONANDIA, I., HOANG, L. K., MAINA, A. S., REGALLA, L. M. & LYONS, T. J. 2008. Heterologous expression of human mPRα, mPRβ and mPRγ in yeast confirms their ability to function as membrane progesterone receptors. Steroids, 73, 1160-1173.

TAKAYANAGI, S., TOKUNAGA, T., LIU, X., OKADA, H., MATSUSHIMA, A. & SHIMOHIGASHI, Y. 2006. Endocrine disruptor bisphenol A strongly binds to human estrogen-related receptor γ (ERRγ) with high constitutive activity. Toxicology Letters, 167, 95-105.

TANG, Y. T., HU, T., ARTERBURN, M., BOYLE, B., BRIGHT, J., EMTAGE, P. & FUNK, W. 2005. PAQR Proteins: A Novel Membrane Receptor Family Defined by an Ancient7-Transmembrane Pass Motif. Journal of Molecular Evolution, 61, 372-380.

VAN DER LINDEN, S. C., HERINGA, M. B., MAN, H.-Y., SONNEVELD, E., PUIJKER, L. M., BROUWER, A. & VAN DER BURG, B. 2008. Detection of Multiple Hormonal Activities in Wastewater Effluents and Surface Water, Using a Panel of Steroid Receptor CALUX Bioassays. Environmental Science & Technology, 42, 5814-5820.

ZHU, Y., BOND, J. & THOMAS, P. 2003. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Science Signaling, 100, 2237.