Magoffin D A
Department of Reproductive Medicine, University of California-San Diego, La Jolla 92093.
Endocrinology. 1989 Sep;125(3):1464-73. doi: 10.1210/endo-125-3-1464.
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.
促黄体生成素（LH）已被证明是调节卵巢卵泡膜间质细胞（TIC）分化的主要激素。已有充分证据表明，LH能刺激环磷酸腺苷（cAMP）的产生，且cAMP类似物可模拟LH的刺激作用，但LH和cAMP刺激TIC分化的机制尚不清楚。这些研究的目的是在无血清培养基中对LH刺激的分离TIC分化进行表征，并研究cAMP依赖性蛋白激酶（PKA）同工酶在TIC分化中的作用。通过Percoll梯度离心从垂体切除的未成熟大鼠经胶原酶分散的卵巢中分离出高度纯化（>90%）且无颗粒细胞污染的TIC。当纯化的TIC（20,000个活细胞/孔）在无血清培养基（96孔板中0.2 ml）中培养（2天）时，会产生低水平的类固醇。LH刺激主要雄激素雄酮产生剂量相关（半数有效剂量[ED50]=2.6±0.4 ng/ml）的增加（50倍）。LH刺激cAMP产生立即呈剂量相关增加，但在LH刺激雄酮产生增加前有20小时的延迟，雄酮产生在30小时达到最高水平。LH刺激的孕酮产生在10小时立即增加到最大值，然后随着雄酮产生增加孕酮水平下降。为确定PKA在刺激雄酮和孕酮产生中的作用，将TIC分别与8-氨基己基氨基-cAMP（100 microM）加N6-苯甲酰-cAMP（30 microM）或8-硫代甲基-cAMP（30 microM）加N6-苯甲酰-cAMP（30 microM）一起培养（2天），以分别直接和选择性地激活I型或II型PKA。任一同工酶的选择性激活均增加了TIC的雄酮和孕酮产生。免疫印迹显示，I型或II型PKA均可增加TIC中细胞色素P450侧链裂解酶（P450scc）和细胞色素P450 17α（P45017α）的含量。这是首次证明直接激活I型或II型PKA均可刺激TIC分化。这些结果表明，LH通过激活一种或两种PKA同工酶介导的机制刺激TIC分化。
