Junction Dynamics and Male Fertility Regulation

描述（由申请方提供）：在精子发生过程中，发育中的生殖细胞必须通过支持细胞和生殖细胞之间的专门锚定连接保持附着在生精上皮上。本项目旨在确定调控生精上皮细胞-细胞肌动蛋白基粘附连接（AJ）的调控分子和信号通路，如外质特化（ES，睾丸特异性AJ型）。如果知道这些信息，就可以操纵和干扰支持细胞和发育中的生殖细胞之间的细胞粘附功能，从而诱导生精上皮中的生殖细胞丢失。由于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-细胞相互作用的位点，并且不干扰下丘脑-垂体-睾丸激素轴。因此，副作用（如果有的话）将是最小的。P.L.的实验室已经利用体内模型来研究AJ动力学，其中使用AF-2364 [1-（2，4-二氯苄基）-吲唑-3-碳酰肼]来扰乱生精上皮中的细胞粘附功能。这些最近的研究表明，支持-生殖细胞AJ在体内的动力学受：（i）整联蛋白/Rho B/ROCK/LIM激酶/cofilin和（ii）整联蛋白/p-FAK/p130 Cas/PI 3-激酶信号通路的调节。现在将使用另一种体内AJ动力学模型，即使用睾酮加雌二醇植入物从上皮细胞中去除雄激素诱导的精子细胞损失（第8步精子细胞及以上），对这些发现进行有力的测试。此外，现代生物化学技术（例如，蛋白质的化学交联、免疫沉淀、免疫印迹），细胞生物学（例如，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（例如，实时RT-PCR、反义ODNs和RNA干扰）将被用于解剖ES和桥粒（一种基于细胞-细胞中间介导的锚定连接类型）的分子结构，并探索信号分子的意义和调节睾丸中细胞粘附的途径。此外，将进一步使用下游蛋白激酶的特异性抑制剂以及建立的体外和体内AJ重组模型来表征位于ES和桥粒位点的多蛋白复合物所利用的信号传导途径。总之，这些研究不仅将确定AJ动力学的调节，还将确定男性避孕药开发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.