ESTABLISHMENT OF A STABLE STEROIDOGENIC PORCINE GRANULOSA-CELL LINE

We report the establishment and preliminary characterization of a stab le steroidogenic granulosa cell line, JC-410. This cell line was obtai ned by spontaneous immortalization of a primary culture of porcine gra nulosa cells. Cultured JC-410 cells produced less progesterone than gr anulosa cells in primary culture. Progesterone synthesis by JC-410 cel ls was approximately 10% and 1% of the amount produced by granulosa ce lls from small and medium sized follicles, respectively. Although FSH and LH did not change progesterone levels in cultured JC-410 cells, fo rskolin and cholera toxin induced a 2.6- and 2.75-fold increase, respe ctively, 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-f old versus control, respectively, after a 24 h incubation. No detectab le levels of estradiol-17 beta were found in JC-410 cells after 48 h i n culture. However, addition of 0.01, 0.1 and 1 mu M androstenedione e levated 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, aromata se and P450scc genes in JC-410 cells is of similar magnitude to the le vel of expression in granulosa cells in primary culture. The JC-410 ce lls have been maintained in culture for more than one year during whic h their population doubled over 100 times. We conclude that JC-410 is a stable cell line that lost responsiveness to the gonadotropins durin g the process of immortalization, but retained its steroid biosyntheti c capability and the expression of key steroidogenic genes. These char acteristics may reflect features of cells arrested in an early stage o f granulosa cell differentiation.