Junction Dynamics and Male Fertility Regulation

描述(申请人提供)：在精子发生过程中，发育中的生殖细胞必须通过Sertoli和生殖细胞之间的特殊锚定连接保持与生精上皮的连接。该项目试图确定调控细胞-细胞肌动蛋白粘连连接(AJ)的调节分子和信号通路，例如生精上皮中的胞质特化(ES，一种睾丸特异的AJ类型)。如果这一信息已知，Sertoli和发育中的生殖细胞之间的细胞黏附功能可以被操纵和干扰，从而导致生精上皮中的生殖细胞丢失。由于在精液中发现的生殖细胞是不成熟的，它们将缺乏使卵子受精的能力，从而导致不育。这种方法专门针对睾丸中细胞-细胞相互作用的部位，不干扰下丘脑-垂体-睾丸激素轴。因此，副作用，如果有的话，将是最小的。P.L.‘S实验室利用体内模型研究了AJ的动力学，使用AF-2364[1-(2，4-dichlorobenzyl)-indazole-3-carbohydrazide]扰乱生精上皮细胞黏附功能。这些最近的研究表明，支持生殖细胞AJ在体内的动态受到：(I)整合素/Rho B/ROCK/Lim激酶/cofilin和(Ii)整合素/p-FAK/p130Cas/PI 3-激酶信号通路的调节。这些发现现在将使用另一个体内AJ动力学模型进行强有力的测试，即使用睾酮加雌二醇植入导致雄激素耗竭导致的上皮精子细胞丢失(第8步精子细胞及以上)。此外，现代生物化学(如蛋白质化学交联、免疫沉淀、免疫印迹)、细胞生物学(如免疫组织化学、免疫荧光和共聚焦显微镜、免疫金电子显微镜)、分子生物学(如实时RT-PCR、反义ODN和RNA干扰)将被用来剖析ES和桥粒(一种基于细胞-细胞中间丝的锚定连接型)的分子结构，并探索信号分子的意义和调节睾丸细胞黏附的途径。此外，驻留在ES和桥粒位置的多蛋白复合体所使用的信号通路将使用下游蛋白激酶的特定抑制剂以及建立的AJ重组的体外和体内模型来进一步表征。总而言之，这些研究不仅将确定AJ动态的调节，还将确定男性避孕发展的新线索和创新方法。

DESCRIPTION (provided by applicant): During spermatogenesis, developing germ cells must remain attached to the seminiferous epithelium via specialized anchoring junctions between Sertoli and germ cells. This project seeks to identify the regulatory molecules and signaling pathways that regulate the cell-cell actin-based adherens junction (AJ), such as the ectoplasmic specialization (ES, a testes-specific AJ type), in the seminiferous epithelium. If this information is known, cell adhesion function between Sertoli and developing germ cells can be manipulated and perturbed, thereby inducing germ cell loss from the seminiferous epithelium. Since germ cells found in the semen are immature, they will lack the ability to fertilize the egg and infertility will result. This approach specifically targets the site of cell-cell interactions in the testes and does not interfere with the hypothalamus-pituitary-testicular hormonal axis. As such, side effects, if any, will be minimal. The P.l.'s laboratory has utilized an in vivo model to study AJ dynamics with the use of AF-2364 [1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide] to perturb cell adhesive function in the seminiferous epithelium. These recent studies have shown that Sertoli-germ cell AJ dynamics in vivo are regulated by: (i) the integrin/Rho B/ROCK/LIM kinase/cofilin and (ii) the integrin/p-FAK/p130 Cas/PI 3-kinase signaling pathways. These findings will now be vigorously tested using another model of in vivo AJ dynamics, namely the androgen depletion-induced spermatid loss (step 8 spermatids and beyond) from the epithelium using testosterone plus estradiol implants. Furthermore, contemporary techniques of biochemistry (e.g., chemical cross-linking of proteins, immunoprecipitation, immunoblotting), cell biology (e.g., immunohistochemistry, immunofluorescent and confocal microscopy, immunogold electron microscopy) molecular biology (e.g., real-time RT-PCR, antisense ODNs, and RNA interference) will be used to dissect the molecular architecture of the ES and desmosome (a cell-cell intermediate filament-based anchoring junction type), and to explore the significance of signaling molecules and the pathways that regulate cell adhesion in the testes. In addition, the signaling pathways utilized by the multiprotein complexes residing at the sites of ES and desmosomes will be further characterized using specific inhibitors of the downstream protein kinases together with established in vitro and in vivo models of AJ restructuring. In summary, these studies not only will define the regulation of AJ dynamics, they will identify new leads and innovative approaches for male contraceptive development.