Research Projects

Project 1 Project 2 Project 3 A beautiful and clean environment!

Project 1 Seasonal Variation in Vitellogenin and Steroid Hormone Levels in Largemouth from Lakes in Central Florida Vitellogenin, the egg yolk precursor protein produced in females, is under the direct control of estradiol 17-b, a form of estrogen present in female fish. The vitellogenin gene is activated in the liver in the fall, just as eggs are beginning to form. Vitellogenin is exported from the liver into the blood stream from where it is picked up by the developing oocyte through receptor-mediated endocytosis. Normal levels of vitellogenin concentration in blood of female largemouth bass during the prime reproductive season reach 10-30 mg/ml. Male fish also have the gene for vitellogenin – but it is normally not active because estradiol levels are below the threshold required for activation. However, if male fish are exposed to estrogen or an estrogen mimic, they become induced and start to produce egg yolk proteins. Because they do not have developing oocytes as a natural sink for the vitellogenin, there is no place for the protein to go. It simply accumulates in the blood until proteases degrade it. The presence of vitellogenin in blood of males is thus an excellent biomarker for exposure to estrogen or estrogen mimics. Both male and female largemouth bass have estrogen and testosterone, what is different between the two are the levels of each hormone. Females have higher estrogen levels, while males have higher testosterone levels. To get a better understanding of how the hormone levels correlate with each other and with vitellogenin synthesis, we have undertaken a seasonal study of largemouth bass. We have measured estradiol, 11-keto testosterone, and vitellogenin levels as a function of the season. We are currently mapping out gene expression in the liver that is under the control of estradiol. Project 2 Differential Display RT-PCR of Laboratory Treated Sheepshead Minnows In this project we are interested in determining the set of genes that are induced by treating male sheepshead minnows with estradiol or estrogen mimics. We have measured vitellogenin induction in the plasma as a function of dose and time. We have prepared total RNA from livers from controls and from treated animals and have compared the mRNAs that are expressed in each. We have found about 50 induced genes which we are currently in the process of identifying. So far, among the mRNAs that we have identified the following are induced: estrogen receptor, vitellogenin, and vitellin envelope proteins. We are in the process of identifying more . Project 3. Development of Monoclonal Antibodies Against Vitellogenin For this project we have developed a library of monoclonal antibodies against fish vitellogenins that cross-react widely. Vitellogenin is broken down into egg yolk proteins in the egg and it is the main nutritional source for the developing embryo. It is not, however, conserved in its primary sequence from one species to another. Thus, normally antibodies made against vitellogenin from one species do not crossreact with that of another. But, vitellogenins do share some similarities in sequence and antibodies that recognize these short sequences do cross react widely. We have developed a series of antibodies that apparently recognized shared epitopes. We have antibodies that work well with largemouth bass, rainbow trout, blue gill sunfish, sheepshead minnow, carp, goldfish, walleye pike, northern pike, fundulus, sturgeon, swordfish, among others. We are in the process of making antibodies to fathead minnows and to birds.