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The role of tubulobulbar complex-endoplasmic reticulum contact sites during junction turnover in the seminiferous epithelium Adams, Arlo Cordell


The focus of this thesis is the characterization of the functions of the membrane contact sites (MCSs) between tubulobulbar complexes (TBCs) and the endoplasmic reticulum (ER). TBCs are double membrane endocytic invaginations that are endemic to Sertoli cells of the mammalian testis. They have been proposed to internalize intact intercellular junctions between late spermatids and Sertoli cells, allowing late spermatids to be released into the seminiferous tubule lumen. However, there is little data showing how TBCs may be regulated temporally and spatially to initiate junction turnover and subsequent spermatid release (spermiation). One of the most striking features of TBCs is the close association of ER to the ‘bulb’ portion, which is present for much of the time a TBC is developing and up until the moment of its vesiculation, all while it is still continuous with the plasma membrane. Although ER contacts are known to help regulate the form and function of other organelles such as endosomes, there have never been an investigation into what the functions of the TBC-ER contact sites are. It is my overall hypothesis that the TBC-ER contact sites are necessary for the normal development of TBCs into mature endocytic structures. Some of the well-established functions of ER contacts are both calcium and lipid exchange. Both processes have been shown to be fundamental to the progression of endosomes through the endosomal-lysosomal system. In this thesis, I begin by using qualitative approaches to establish that proteins known to be involved in both calcium and lipid exchange are present at TBC-ER contacts. I follow-up on my qualitative works with functional studies where I explore the role some of these proteins play in TBC development and in spermiation. Using in vivo knockdown experiments in rat testes to target two of these proteins (IP3R1 and ORP9), I demonstrate that both are necessary for the normal maturation of TBC bulbs and for sperm release to occur. These data support that conclusion that both calcium and lipid exchange are functions of the TBC-ER contact sites and provide evidence that the contact sites are necessary components of the sperm release mechanism.

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