Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen

Studies of human ovarian granulosa cells have been limited by the small numbers and short life span in culture of cells currently obtainable from clinical material. Using SV40 large T antigen, we have reproducibly immortalized freshly explanted human granulosa cells obtained through an In Vitro Fertilization Program. Of 69 independently isolated clones, 17 grew progressively into lines. In the presence of 1 mM 8-Br-cyclic AMP and 50 ng/ml pregnenolone, 15 of these 17 lines secreted progesterone. Seven lines derived from two patients were analyzed in detail. The lines differed in their responsiveness to cyclic AMP and pregnenolone, and in the timing of their steroidogenic responses. In response to cyclic AMP plus pregnenolone, progesterone secretion increased up to 20-fold. Three clones tested all responded to forskolin and cholera toxin with up to 7-fold increases in progesterone secretion. One line responded inconsistently to 1 IU/ml hCG but not to FSH. The steroidogenic responses to cyclic AMP were accompanied by morphologic cell rounding. The immortalized cell lines underwent 40-60 population doublings, thus, providing a theoretical yield of up to 10(18) cells per line. These results show that a high proportion of SV40 immortalized lines is steroidogenic. These lines provide a new experimental model for studies of cell lines that appear representative of different states of differentiation of human granulosa cells.