Establishment and characterization of a primary culture of ovarian follicular cells for sea bass (Dicentrarchus labrax)

Resumen del trabajo presentado en el 25th Conference of European Comparative Endocrinologists, celebrada en Pécs (Hungría), del 31 de agosto al 4 de septiembre de 2010Follicular somatic cells support oocyte development, where interactions between the germ cells and the surrounding follicular cells are critical for the successful maintenance of the reproductive activity. The absence of immortalized granulosa or theca cell lines in fish led us to develop a primary culture system that enabled in vitro studies of processes that take place within the ovary along the reproductive cycle. For this purpose, we optimized the isolation procedure as well as the culture conditions of sea bass ovarian follicular cells, and evaluated their behaviour at three different physiological temperatures (25°C, 18°C and 15°C). To characterize this primary culture, the steroidogenic capability of the follicular cells was measured periodically along 16 days. For this aim, culture medium was collected every 4th day and levels of both testosterone and estradiol were determined through specific EIAs. Moreover, expression of several genes, typically characteristic of granulosa and theca cells, was tested to ensure that the cellular types present in the culture were those of the follicular layer. Finally, to evaluate the suitability of this system for gene function studies several transient transfection conditions were tested. As a first qualitative approach, an expression vector containing the green fluorescent protein (GFP) under the control of the human cytomegalovirus promoter (CMV) was introduced into the cells through different transfection reagents: Lipofectamine (Invitrogen Corp), Fugene and Fugene 6 (Roche Diagnostics) and the conventional calcium phosphate method. GFP expression was monitored by means of a fluorescence microscope. The most efficient transfection methods (Fugene 6 and calcium phosphate) were verified by transfection of different plasmids containing the luciferase gene, and subsequent quantification of the luciferase activity. All together our results show that this primary culture could be a good in vitro model to study follicular cell physiology in fish, and a useful conspecific expression system for functional analysis of certain sea bass genes and promoters related with ovarian development.Research supported by MEC (2006 3 O1 037) and MICINN (AGL2008-02937