LH has been shown to be the principal
hormone regulating ovarian thecal-interstitial cell (
TIC) differentiation. It has been well documented that LH stimulates cAMP production and that cAMP analogs mimick the stimulatory actions of LH, but the mechanisms by which LH and cAMP stimulate
TIC differentiation are unknown. The purpose of these studies was to characterize LH-stimulated differentiation of isolated
TIC in serum-free medium and examine the role of
cAMP-dependent protein kinase (PKA)
isoenzymes in
TIC differentiation. Highly purified (greater than 90%)
TIC which were free from granulosa cell contamination were isolated from
collagenase-dispersed ovaries of hypophysectomized immature rats by
Percoll gradient centrifugation. When the purified
TIC (20,000 viable cells/well) were cultured (2 days) in serum-free medium (0.2 ml in 96-well plates), low levels of
steroids were produced. LH stimulated a dose-related (ED50 = 2.6 +/- 0.4 ng/ml) increase (50-fold) in
androsterone, the principal
androgen produced. LH stimulated an immediate dose-related increase in cAMP production, but there was a 20-h lag before LH stimulated an increase in
androsterone production, which reached maximum levels at 30 h. LH-stimulated
progesterone production increased immediately to a maximum
at 10 h, then
progesterone levels decreased as
androsterone production increased. To determine the role of PKA in stimulating
androsterone and
progesterone production,
TIC were cultured (2 days) with 8-aminohexylamino-cAMP (100 microM) plus N6-benzoyl-cAMP (30 microM) or
8-thiomethyl-cAMP (30 microM) plus N6-benzoyl-cAMP (30 microM) to directly and selectively activate type I or type II PKA, respectively. Selective activation of either
isoenzyme increased
androsterone and
progesterone production by
TIC. Immunoblots revealed that either type I or type II PKA increased the contents of P450scc and P45017 alpha in
TIC. This is the first demonstration that direct activation of either type I or type II PKA stimulates
TIC differentiation. These results indicate that LH stimulates
TIC differentiation by a mechanism mediated by activation of one or both PKA
isoenzymes.