Junction Dynamics and Male Fertility Regulation

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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.