Physiology and Endocrinology II

Activated estrogen receptors (ER) in response to estrogen bind to specific sequences (estrogen response elements; ERE) to induce transcription. The objective of this study was to evaluate whether the estrogen induced ER binding activity in granulosa cells of antral ovarian follicles can be detected by bioluminescence imaging in vitro, and correlated to estrogen concentrations in follicular fluid. In this study, we used lipid–mediated gene transfer and an ERE–luc reporter gene (which consisted of 3 tandem repeats of EREs upstream from the luciferase gene) to transfect granulosa cells within intact follicles. When the endogenous functional and activated ERs bind to the ERE–luc sequences within the transfected granulosa cells, the expression of luciferase is enacted for detection. A total of n = 58 follicles between 4.2 to 9.4 mm in diameter were dissected from the ovaries. DNA– lipid complexes were formed at a DNA (μg): lipid (μl) ratio of 1:3, by adding FuGene 6 in PBS to 3 μg of ERE–luc DNA and injected into each follicle using a microinjector. The follicles were cultured individually with αMEM and 45% O2; 50% N2; 5% CO2 at 39°C. After 20 h post–transfection, the luminescence from each follicle was detected using an IVIS 100 imaging system. Each follicle was imaged with 10 min exposure and signal intensity was reported (and normalized) as mean ± SEM of photons per second (p/s). Estradiol concentrations of follicular fluid were measured by radioimmunoassay in each follicle. Regression coefficients were determined, and a P–value of <0.05 was considered significant. Concentrations of estradiol in follicular fluid significantly increased as follicle size increased (r = 0.607; P < 0.05). Estradiol total content in each follicle was positively correlated with ERE-driven luciferase expression level of follicles (r = 0.39; P < 0.05). These results demonstrate the initial development of a new methodology for measuring functional and ligand activated estrogen receptor activity within intact porcine ovarian follicles in vitro using bioluminescence imaging methodologies.