Koduri Sailaja, Hild Sheri Ann, Pessaint Laurent, Reel Jerry R, Attardi Barbara J
Division of Reproductive Endocrinology and Toxicology, BIOQUAL Inc., 9600 Medical Center Drive, Rockville, MD 20850-3336, USA.
Endocrinology. 2008 Apr;149(4):1850-60. doi: 10.1210/en.2007-1332. Epub 2008 Jan 3.
The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study 45-d-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIAs or ELISAs, and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-beta1, N-cadherin, alpha-catenin, and beta-catenin protein at 24 h and later time points. Increase in expression of Fas ligand and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022-treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of stem cell factor, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.
本研究旨在阐明茚并吡啶类化合物l-CDB-4022抗生精活性可能涉及的分子机制。在本研究中，给45日龄雄性Sprague-Dawley大鼠单次口服l-CDB-4022（2.5 mg/kg）或赋形剂，并在不同时间点采集血液和睾丸。体重增加率未受影响，但l-CDB-4022导致睾丸重量显著减轻。使用特异性放射免疫分析法（RIAs）或酶联免疫吸附测定法（ELISAs）检测血清激素，分别通过蛋白质免疫印迹法和逆转录聚合酶链反应（RT-PCR）分析睾丸蛋白和RNA。l-CDB-4022处理组大鼠血清中抑制素B显著降低，促卵泡生成素（FSH）相应升高，但激活素A、睾酮和促黄体生成素（LH）的血清水平未变。对l-CDB-4022处理组大鼠睾丸裂解物进行的蛋白质免疫印迹分析显示，在4小时及之后的时间点细胞外信号调节激酶1/2（ERK1/2）发生磷酸化。48小时时nectin/afadin复合物消失，但在24小时及之后的时间点整合素β1、N-钙黏蛋白、α-连环蛋白和β-连环蛋白的蛋白水平升高。l-CDB-4022处理8小时和24小时后，检测到Fas配体和Fas受体的表达增加。干细胞因子mRNA的膜结合型与可溶性形式的比例降低。对睾丸切片进行的免疫组织化学分析表明，l-CDB-4022处理组大鼠的支持细胞微管网络遭到严重破坏。总体而言，这些结果表明，l-CDB-4022激活丝裂原活化蛋白激酶（MAPK）途径，降低诸如膜结合型干细胞因子等促生存因子的表达，改变支持细胞-生殖细胞黏附连接蛋白的表达，破坏支持细胞微管结构，并诱导促凋亡因子Fas表达，最终导致生精上皮中的生殖细胞丢失。
