|Jung, Song-Yi -|
|Willard, Scott -|
Submitted to: Reproductive Biology and Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 19, 2014
Publication Date: January 30, 2014
Citation: Jung, S., Willard, S.T. 2014. Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro. Reproductive Biology and Endocrinology. 12:11. Interpretive Summary: The ovarian antral follicle consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. In this study, we explored the a new imaging tool which is based on light-emitting gene transcription and have applied this to intact living antral follicles from pig ovaries. We have demonstrated a dose response to get optimal light-emitting (via luciferase) gene expression, and the effect of the enzyme substrate (D-luciferin) amount on emission characteristics in this system. This methodology can be used to determine specific levels of functional gene expression during the oocyte maturation process within the follicle and possibly applied to gene therapy approaches to improve oocyte maturation and quality.
Technical Abstract: The porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles. We analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 µg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro. The level of luciferase activity of follicles with 3 µg pGL4 was significantly (P'<'0.05) greater than the 1 µg and 2 µg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P'<'0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 µg of D-luciferin. The results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles.