Abstract
The work presented in this dissertation employs the use of de novo formation of seminiferous tubules in vivo and in vitro to study the testicular microenvironment, with a focus on the role of Vascular Endothelial Growth Factor A (VEGFA) and the primary cilium organelle. First, we optimized the de novo morphogenesis assay by testing different methods to improve the spermatogenic efficiency of the bioassay. We showed that addition of Matrigel to the cell pellet prior to grafting results in a better efficiency than enrichment of germ cells, and when cells are grafted under the same conditions, the number of cells transplanted directly relates to the number of seminiferous tubules formed. Second, we evaluated the role of VEGFA on the reestablishment of the testicular microenvironment. We showed that de novo formed tubules developed from cell pellets treated with VEGFA contained higher number of proliferating cells per tubule and higher spermatogenic efficiency. These findings suggest that VEGFA has a protective role during the grafting process. Another study explored the stirred suspension bioreactor (SSB) as a novel method to enrich for germ cells on a large scale. We used the optimized de novo morphogenesis assay to functionally assess the physiological abilities of cells cultured under shear force stress in the SSB. Results of these experiments indicate that SSB is a practical alternative to attain enriched populations of germ cells on a large scale, which maintains the functional spermatogenic potential of the cells. Finally, the last chapter focused on the role of the primary cilium in the testicular microenvironment. This set of experiments used the small molecule inhibitor, Ciliobrevin D, to disturb the organelle. We demonstrated that lack of primary cilia blocks Hedgehog signaling and impairs tubule formation in vitro. Overall, the work presented in this dissertation contributes to the improvement of available models to study male reproductive biology and to our understanding of the role of VEGFA and primary cilia in the testicular microenvironment.
