Fournet N, Weitsman S R, Zachow R J, Magoffin D A
Department of Obstetrics and Gynecology, Cedars-Sinai Research Institute/University of California, Los Angeles School of Medicine 90048, USA.
Endocrinology. 1996 Jan;137(1):166-74. doi: 10.1210/endo.137.1.8536609.
Transforming growth factor-beta1 (TGF-beta1) inhibits theca-interstitial cell (TIC) androgen biosynthesis while enhancing progesterone production without altering P45017 alpha protein content. The purpose of the present study was to define the mechanism of TGF-beta 1 inhibition of ovarian androgen production by determining the effects of TGF-beta 1 on steroidogenic enzyme messenger RNA (mRNA) expression and 17 alpha-hydroxylase activity in TIC in vitro. TIC isolated from hypophysectomized immature rat ovaries by Percoll gradient centrifugation were cultured with and without LH and TGF-beta 1 up to 6 days. At various times, cytoplasmic mRNA was extracted from the TIC, and P450scc, 3 beta-HSD and P450(17 alpha) mRNA were measured by specific assays, using RT-PCR. Treatment with TGF-beta 1 alone (0.1-100 ng/ml) had no effect on mRNA expression at 2 days but increased P450scc and 3 beta-HDS mRNA at 4 days. TGF-beta did not alter the LH stimulation of P450scc and 3 beta-HSD mRNA up to 6 days but caused a modest (2.5-fold) increase in P450 (17 alpha) mRNA at 2 days. Specificity studies with inhibin-A (30 ng/ml), activin-A (100 ng/ml), and MIS (300 ng/ml) demonstrated that the effects of TGF-beta 1 were unique within this family of peptides. We next examined the effect of TGF-beta 1 on 17 alpha-hydroxylase activity. Kinetic analysis revealed that the 17 alpha-hydroxylase enzyme has an apparent Michaelis-Menten constant of 3.42 mumol/liter and maximum velocity of 0.23 pmol/min x mg protein. TGF-beta 1 inhibited 17 alpha-hydroxylase activity by a noncompetitive mechanism with an apparent inhibin constant (Ki) of 46.4 pM. The results of our studies demonstrate that TGF-beta 1 directly inhibits TIC androgen production by a noncompetitive mechanism. This novel mechanism may be important in preventing excessive androgen production in developing ovarian follicles without preventing differentiation of the TIC.
转化生长因子-β1（TGF-β1）可抑制卵泡膜间质细胞（TIC）雄激素的生物合成，同时增加孕酮的生成，且不改变P45017α蛋白的含量。本研究的目的是通过测定TGF-β1对体外培养的TIC中类固醇生成酶信使核糖核酸（mRNA）表达及17α-羟化酶活性的影响，来明确TGF-β1抑制卵巢雄激素生成的机制。通过Percoll梯度离心法从垂体切除的未成熟大鼠卵巢中分离出TIC，并在添加或不添加促黄体生成素（LH）和TGF-β1的条件下培养长达6天。在不同时间点，从TIC中提取细胞质mRNA，并使用逆转录聚合酶链反应（RT-PCR）通过特异性检测方法测定细胞色素P450侧链裂解酶（P450scc）、3β-羟基类固醇脱氢酶（3β-HSD）和P450（17α）mRNA的水平。单独使用TGF-β1（0.1 - 100 ng/ml）处理在2天时对mRNA表达无影响，但在4天时可增加P450scc和3β-HDS mRNA的水平。在长达6天的时间里，TGF-β未改变LH对P450scc和3β-HSD mRNA的刺激作用，但在2天时使P450（17α）mRNA适度增加（2.5倍）。使用抑制素-A（30 ng/ml）、激活素-A（100 ng/ml）和苗勒管抑制物质（MIS，300 ng/ml）进行的特异性研究表明，TGF-β1在该肽家族中具有独特的作用效果。接下来，我们检测了TGF-β1对17α-羟化酶活性的影响。动力学分析显示，17α-羟化酶的表观米氏常数为3.42 μmol/升，最大反应速度为0.23 pmol/分钟×毫克蛋白。TGF-β1通过非竞争性机制抑制17α-羟化酶活性，其表观抑制常数（Ki）为46.4 pM。我们的研究结果表明，TGF-β1通过非竞争性机制直接抑制TIC雄激素的生成。这一全新机制对于在不阻止TIC分化的情况下，防止发育中的卵巢卵泡过度生成雄激素可能具有重要意义。
