Abstract
BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsThe 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.
Highlights
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
Luciferase activity of intact follicles did not differ between the 2 μg and 1 μg groups (P = 0.62) at 1 min after luciferin injection
In the present study, we have demonstrated the development of a new methodology for measuring luciferase transgene expression in porcine antral follicles in vitro using an intact follicle culture system and bioluminescence imaging
Summary
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. The ovarian antral follicle consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid It is an essential microenvironment for oocyte maturation and its developmental competence; reviewed by Moor et al [1] and Hunter [2]. With recent advances in gene transfer methods, many laboratories have used reporter gene technologies to study functional gene activity and transcription factor interactions in signal transduction pathways involved in follicular atresia and steroidogenesis in cultured granulosa cells in vitro [6,7,8,9] These studies have offered the opportunity to elucidate gene functions and cellular pathways within cultured granulosa cells, it does not take into account major interactions between the oocyte and the follicular components in the context of the whole follicle. In this study, we determine the potential to develop a new methodology to measure changes in cellular and molecular events during the oocyte maturation in the context of the living intact follicular environment
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