Establishment of a stable steroidogenic porcine granulosa cell line

We report the establishment and preliminary characterization of a stable steroidogenic granulosa cell line, JC-410. This cell line was obtained by spontaneous immortalization of a primary culture of porcine granulosa cells. Cultured JC-410 cells produced less progesterone than granulosa cells in primary culture. Progesterone synthesis by JC-410 cells was approximately 10% and 1% of the amount produced by granulosa cells from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, forskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respectively, versus control. The JC-410 cells responded to 0.1, 1 and 5 mM cAMP with an increase in progesterone synthesis of 2.5-, 28- and 49-fold versus control, respectively, after a 24 h incubation. No detectable levels of estradiol-17 beta were found in JC-410 cells after 48 h in culture. However, addition of 0.01, 0.1 and 1 mu M androstenedione elevated the levels of estradiol-17 beta to 0.028, 0.3 and 1.21 pg/mu g protein, respectively. The level of expression of 3 beta-HSD, aromatase and P450scc genes in JC-410 cells is of similar magnitude to the level of expression in granulosa cells in primary culture. The JC-410 cells have been maintained in culture for more than one year during which their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins during the process of immortalization, but retained its steroid biosynthetic capability and the expression of key steroidogenic genes. These characteristics may reflect features of cells arrested in an early stage of granulosa cell differentiation.