Intercellular junction disassembly : assigning a function to apical tubulobulbar complexes in testis

The focus of this thesis is the characterization of apical tubulobulbar complexes in mammalian testis. These double-membrane, actin-based structures form at sites of attachment between germ cells and Sertoli cells. The location, timing and morphology of these complexes have inspired several proposed functions. It has been proposed that tubulobulbar complexes serve as an anchor to prevent early release of spermatids from the epithelium or that they are a device for elimination of excess cytoplasm. I hypothesize that tubulobulbar complexes are subcellular machines responsible for the internalization of intact intercellular junctions thereby contributing to the process of spermatid release from the seminiferous epithelium. A descriptive approach is taken to determine if key components that are present at similar structures in other systems also are present at tubulobulbar complexes, to determine if integral junction molecules are present at tubulobulbar complexes and to determine the fate of internalized junction material. A functional approach is taken to deplete the expression of an actin-based protein that is localized to tubulobulbar complexes to test the prediction that the structures are involved with spermatid release. Dendritic actin components were localized around the cuff of tubulobulbar complexes and clathrin was localized to coated pits at the ends of the structures. Based on these data, a model of tubulobulbar complex formation was proposed that incorporates clathrin-mediated endocytosis and dendritic actin assembly. Junction molecules known to be present at ectoplasmic specializations were found in vesicles at the ends of tubulobulbar complexes that label positively for early endosome markers. Interestingly, junction protein nectin 2 was colocalized with recycling marker Rab11 at newly forming junctions deeper in the epithelium. This suggests that recycling of junctional proteins may be occurring in Sertoli cells. Finally, depletion of cortactin, a key protein at tubulobulbar complexes, resulted in a short phenotype – an indication that the structures were not able to acquire or maintain their normal length after treatment. Significantly, delay in spermatid release was detected. The data presented here supports the junction internalization hypothesis and introduces a new paradigm for junction internalization generally in cells and links the mechanism to a biologically significant event – sperm release.Medicine, Faculty ofGraduat

Cortactin depletion results in short tubulobulbar complexes and spermiation failure in rat testes

Summary Tubulobulbar complexes are actin-related endocytic structures that form at sites of intercellular attachment in the seminiferous epithelium and are proposed to internalize intact junctions. In this study, we test the prediction that altering the structure/function of tubulobulbar complexes results in failure to release mature spermatids from Sertoli cells. We used an in vivo knockdown strategy to target cortactin, a component of tubulobulbar complexes, in Sprague Dawley rats. In each animal, one testis was surgically injected with cortactin siRNA reagents and the other testis was injected with non-targeting siRNA. After three days, experimental and control testes were processed for immunoblotting, electron microscopy or immunofluorescence microscopy. In testis sections immunostained for cortactin or labeled for filamentous actin, fluorescence microscopy revealed that tubulobulbar complexes were shorter in siRNA-treated testes relative to controls. Significantly, in the knockdown testes, spermiation was delayed in some tubules and had failed in others. When evaluated by electron microscopy, adhesion complexes (ectoplasmic specializations) remained associated with mature spermatids that failed to be released from Sertoli cells. Immunoblots both of whole testis lysates and of isolated seminiferous epithelial lysates confirmed that cortactin expression was knocked-down in experimental testes and in the seminiferous epithelium respectively, relative to controls. Moreover, in testes injected with siRNA reagents with a dye modification on one of the four targeting siRNA sequences, dye clusters were detected at the base of the epithelium confirming that the reagents entered Sertoli cells. Our results are consistent with the hypothesis that tubulobulbar complexes internalize intercellular junctions and that they are a significant component of the sperm release mechanism