Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Abstract

We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 M of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20␣-hydroxy-4-pregnen-3-one (20␣-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMPinduced progesterone synthesis only moderately. Estradiol, insulinlike growth factor I, and insulin showed no significant effect on cAMPinduced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK-and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochrondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK-or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells. (Endocrinology 139: 4679 -4687, 1998)

Directed Mouse Embryonic Stem Cells into Leydig-Like Cells Rescue Testosterone-Deficient Male Rats In Vivo

“…These results are consistent with earlier reports [41][42][43]. Erik Willems et al suggested that FSK acted on the expression of SF-1 mRNA at the transcriptional level, and this was determined to be possible because FSK-induced enhanced expression of SF-1 mRNA was almost completely abolished by actinomycin D [39].…”

Contribution of progesterone, follicle stimulating hormone and glucocorticoids in survival of serum-free cultured granulosa cell explants

“…Although it is known that glucocorticoids highly augment cAMP-stimulated progesterone production in several granulosa models (Hosokawa et al 1998), its regulatory role in survival is not well documented (Makrigiannakis et al 2000).…”

Androgen excess down-regulates connexin43 in a human granulosa cell line

“…The HO23 human immortalized luteinized granulosa cell line was provided by Dr. Abraham Amsterdam, Weizmann Institute of Science, Rehovot, Israel (13). Granulosa cells were maintained at 37 C in 5% CO 2 /air in Dulbecco's minimal essential medium (DMEM)/Ham's F12 (1:1), supplemented with 5% fetal calf serum and antibiotics (100 IU/mL of penicillin and 100 mg/mL of streptomycin).…”