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Title:In vitro model of the fathead minnow hypothalamic-pituitary-gonadal axis for toxicological evaluation
Author(s):Johnston, Theresa
Director of Research:Cropek, Donald M.
Doctoral Committee Chair(s):Ferguson, Duncan C.
Doctoral Committee Member(s):Cropek, Donald M.; Flaws, Jodi A.; Mahoney, Megan M.; Kong, Hyun Joon
Department / Program:Comparative Biosciences
Discipline:VMS - Comparative Biosciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Endocrine disruption
tissue culture
fathead minnow
in vitro toxicology
Abstract:Endocrine disrupting compounds (EDCs) are prevalent in the environment and affect both animals and humans. They cause serious concerns for individuals and populations because they impact reproduction by interfering with the hypothalamic-pituitary-gonadal axis (HPG axis). This changes fecundity at the organismal and population level, potentially leading to population crashes. Development of chemicals for agricultural, industrial, and personal use has led to over 80,000 chemicals with unknown endocrine action. Numerous in vivo and in vitro testing methods have been developed to screen these chemicals. However, extrapolation of in vitro studies to in vivo action is not always accurate. The aim of this research were to develop a tissue co-culture of the fathead minnow (Pimphales promelas) HPG axis and liver to serve as an in vitro system that reproduces the interaction of the fathead minnow HPG axis in vivo. This model system was tested by evaluating the effects of a known EDC, trenbolone acetate (TRB), with the hypothesis that the responses would correlate with those of the HPG in an intact fish. Brain (hypothalamus), pituitary, gonad, and liver tissue were removed from euthanized adult male and female fathead minnow and sliced. Slices were exposed to various culture treatments including media type, serum type, incubation temperature, pH, culture surface, and media change frequency. Tissue viability was measured at 7 or 28 days for each experiment using the MTT assay for cell viability. A culture was considered successful when tissue viability averaged 80% or higher by the end of the culture period. It was hypothesized that the HPG-L tissues would maintain ≥80% viability after 28 days in culture if a combination of media and conditions including temperature, pH, substrate, and handling time were selected for highest tissue viability. All tissues were subsequently cultured and evaluated in this well-defined combination of selected iii conditions. Hormone treatments were used to test function of the tissues by adding hormones to media weekly during a 28 day culture. Liver was dosed with 208 pg/mL estradiol (E2), the sex steroid that stimulates vitellogenin (Vtg) production in egg producing animals. Testes and ovaries were dosed with 5 IU pregnant mare serum gonadotropin (PMSG), which contains gonadotropins that stimulate production of sex steroids. It was hypothesized that E2 would stimulate Vtg production in the liver over 28 days and PMSG would stimulate 11-ketotestosterone (11-KT) production in testes and E2 production in the ovaries, but all hormone production would likely decrease from day 0 of the culture to day 28. Additionally, tissues were exposed to three different concentrations of these hormones in a 24h culture to evaluate the dose-response of each tissue type to its stimulant. After development of culture conditions, tissues were tested for their ability to communicate with each other in co-culture and in separate cultures when exposed to shared media. Ovary tissues were cultured individually and then stimulated with media from brain-pituitary co-cultures mixed with fresh media. It was hypothesized that ovaries would produce E2 in a dose-response manner depending upon the proportion of media they received from the brain-pituitary culture. As the proportion of media from the brain-pituitary cultured increased, the gonadotropin concentration should be greater, increasing stimulation of E2 production. Liver was cultured in three individual treatments and in combination with brain and ovary. Liver in individual treatments was exposed to fresh media, 50% media from ovary individually cultured mixed with 50% fresh media, and 50% media from ovary co-cultured with brain and pituitary mixed with 50% fresh media. It was hypothesized that individual liver cultures exposed to media from the ovary, brain, and pituitary co-culture would produce the largest amount of Vtg because all iv components of the HPG-axis are present and able to function as a system while individual liver cultures exposed only to fresh media would produce the smallest amount of Vtg. After it was shown that the tissues were able to interact in vitro, all four HPG-L tissues were co-cultured each for male and female FHM. Co-cultures were exposed to TRB, a known androgenic EDC, at concentrations similar to those found in tissues after exposure in an in vivo study. Additionally, individual testicular and ovarian tissues were exposed to the same concentrations. E2, 11-KT, and Vtg were measured in these samples. It was hypothesized that the co-culture would respond to the EDC in a similar fashion to the in vivo study while results with the individually cultured gonads do not. Both male and female HPG-L cultures responded to TRB with changes in hormone concentrations that resembled those of a previously reported in vivo study (Ankley et al. 2003). After three days in culture, male HPG-L tissues responded to the three concentrations of TRB with changes in E2, testosterone T, and Vtg with the same trend in hormone changes as the in vivo research. In contrast, the E2 production of testis cultures was similar to the in vivo study in response to the TRB, while the production of other hormones was not. These data suggest that a co-culture system with all components of the HPG-L axis represents in vivo responses better than individual gonads. This system can be enhanced with improvements that focus on better modeling and replicating the in vivo environment. Hydrogel biomaterials can serve as a matrix for tissues that resembles the extra-cellular matrix when chemical, physical and mechanical properties, such as adhesion strength, porosity, and stiffness, are matched to the cell environment in each tissue. For instance, both electromechanical and acoustic radiation force optical coherence tomography were v used to measure the elastic modulus of fathead minnow tissues. Due to the small size of the tissues, only liver and ovary were measurable. Alginate hydrogel was cured using 40 μL/mL, 60 μL/mL, and 80 μL/mL CaSO4 solution to create gels with different elastic modulus. These gels had an elastic modulus that increased with increasing CaSO4 concentration. HPG-L tissues were embedded in each of the alginate gels to determine impact of mechanical stiffness on viability. However, all tissues, including controls had low viability by the end of the culture period and the study was inconclusive but promising. A microfluidic device was designed as a second method of improving cellular environment and tissue interaction by enclosing tissues in individual compartments and flowing media among them. Culture studies have yet to be conducted using this device, but it is promising as a technique for future co-culture studies with this system. The fathead minnow HPG-L culture system is an organ system that can be cultured to maintain viability and function for both acute and chronic tests. This research challenged the system with an acute test, in which the culture did respond to a known EDC similarly to the in vivo response. This demonstrates that this is a responsive system that should be studied further for understanding the HPG-L interactions and responses to EDCs.
Issue Date:2014-01-16
Rights Information:Copyright 2013 Theresa Johnston
Date Available in IDEALS:2014-01-16
Date Deposited:2013-12

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