Erickson G F, Li D, Shimasaki S, Ling N, Weitsman S R, Magoffin D A
Department of Reproductive Medicine, University of California, San Diego, 92093-0674, La Jolla, California.
Endocrine. 1995 Jul;3(7):525-31. doi: 10.1007/BF02738828.
There has been considerable interest in rat ovarian insulin-like growth factor binding proteins IGFBPs because they are potent inhibitors of FSH action.In situ, IGFBP-2 and -4 and IGFBP-3 mRNAs are expressed in rat theca interstitial (TIC) and theca lutein cells respectively. Although much is known about IGFBPs in rat TIC at the mRNA level, the synthesis and regulation of IGFBP proteins remain poorly understood. The purpose of this study was to identify the species of IGFBPs produced by TIC and to determine the effects of LH and IGF-1 on their expression. This was accomplished by culturing rat TIC for 2 days in serum-free medium with graded doses of LH and/or IGF-I, and measuring IGFBP mRNAs in the cells and IGFBP proteins in the conditioned media by RT-PCR and Western immunoblotting respectively. The RT PCR analysis identified strong bands for IGFBP-2 and -4 mRNAs in TIC. In some treatments, the mRNAs for IGFBP-3 and -6 were also identified, but transcripts for IGFBP-1 and -5 were undetectable. Two species of IGFBPs were detected in the conditioned media of control (untreated) TIC, the 31 kDa IGFBP-2 and the 24 kDa (non-glycosylated) and 28 kDa (glycosylated) forms of IGFBP-4. There was no detectable IGFBP-5 and barely detectable amounts of IGFBP-3 and -6 in the conditioned media. Treatment with LH (0.2-20 μU/ml) caused no significant changes in the levels of the 31 kDa IGFBP-2 and the 24 kDa and 28 kDa IGFBP-4 bands, and there was no detectable IGFBP protease activity. In contrast, IGF-I (100 ng/ml) stimulated the expression of IGFBP-2, IGFBP-4 and a 17.5 kDa IGFBP-4 fragment. The immunoreactive IGFBP-4 fragment suggests the media contained an IGFBP-4 protease. The IGF-I effects were dose dependent (ED(50)=12.4±3.3 ng/ml). Co-treating TIC with LH (0.2-20 μU/ml) caused no significant change in the activity of IGF-I in stimulating the expression of IGFBP-2, IGFBP-4 and IGFBP-4 protease. We have demonstrated that IGF-I acts directly on rat TIC to stimulate the expression of the intrinsic IGFBP system. LH, either alone or together with IGF-I, did not significantly change the expression of TIC IGFBP proteins. Therefore, we hypothesize that IGF-I, but not LH, may be a physiologically important regulator of the IGFBP system in rat TIC. Because IGF-I is a potent stimulator of theca function, changes in the expression of this intrinsic IGFBP system could have new implications for ovarian androgen production, both at the physiologic and pathophysiologic levels.
大鼠卵巢胰岛素样生长因子结合蛋白（IGFBPs）一直备受关注，因为它们是促卵泡激素（FSH）作用的强效抑制剂。在大鼠卵泡膜间质细胞（TIC）和黄体膜细胞中，IGFBP - 2、 - 4以及IGFBP - 3的信使核糖核酸（mRNA）分别原位表达。虽然在mRNA水平上对大鼠TIC中的IGFBPs已有很多了解，但IGFBP蛋白的合成与调控仍知之甚少。本研究的目的是鉴定TIC产生的IGFBPs种类，并确定促黄体生成素（LH）和胰岛素样生长因子-1（IGF - 1）对其表达的影响。这是通过在无血清培养基中用不同剂量的LH和/或IGF - I培养大鼠TIC 2天，并分别通过逆转录聚合酶链反应（RT - PCR）和蛋白质免疫印迹法检测细胞中的IGFBP mRNA和条件培养基中的IGFBP蛋白来实现的。RT - PCR分析在TIC中鉴定出了IGFBP - 2和 - 4 mRNA的强条带。在某些处理中，还鉴定出了IGFBP - 3和 - 6的mRNA，但未检测到IGFBP - 1和 - 5的转录本。在对照（未处理）TIC的条件培养基中检测到了两种IGFBPs，即31 kDa的IGFBP - 2和24 kDa（非糖基化）及28 kDa（糖基化）形式的IGFBP - 4。在条件培养基中未检测到IGFBP - 5，IGFBP - 3和 - 6的含量也几乎检测不到。用LH（0.2 - 20 μU/ml）处理后，31 kDa的IGFBP - 2以及24 kDa和28 kDa的IGFBP - 4条带水平没有显著变化，且未检测到IGFBP蛋白酶活性。相比之下，IGF - I（100 ng/ml）刺激了IGFBP - 2、IGFBP - 4和一个17.5 kDa的IGFBP - 4片段的表达。具有免疫反应性的IGFBP - 4片段表明培养基中含有一种IGFBP - 4蛋白酶。IGF - I的作用具有剂量依赖性（半数有效剂量[ED(50)] = 12.4±3.3 ng/ml）。用LH（0.2 - 20 μU/ml）与TIC共同处理，在刺激IGFBP - 2、IGFBP - 4和IGFBP - 4蛋白酶表达方面，IGF - I的活性没有显著变化。我们已经证明，IGF - I直接作用于大鼠TIC以刺激内源性IGFBP系统的表达。单独或与IGF - I一起使用的LH并没有显著改变TIC中IGFBP蛋白的表达。因此，我们推测IGF - I而非LH可能是大鼠TIC中IGFBP系统的生理重要调节因子。由于IGF - I是卵泡膜功能的强效刺激剂，这种内源性IGFBP系统表达的变化可能在生理和病理生理水平上对卵巢雄激素的产生具有新的意义。
