Tubulobulbar complexes (TBCs) are actin-related endocytic structures that internalize intercellular junctions in the seminiferous epithelium. The structures consist of elongate tubular projections of the attached plasma membranes of two adjacent cells that project into Sertoli cells. This double membrane core is cuffed by a dentritic actin network and is capped at its end by a clathrin-coated pit. Here we explore the possibility that elements of the spectrin cytoskeleton are associated with clusters of tubulobulbar complexes that develop at adhesion junctions between late spermatids and Sertoli cells at the apex of the epithelium, and extend what is known about the distribution of plectin at the sites.Cryo-sections of perfusion-fixed testes and apical processes of Sertoli cells mechanically dissociated from perfusion-fixed testes were probed for spectrin, EPB41, and actin and analyzed using conventional fluorescence microscopy and confocal microscopy. Data sets from confocal microscopy were analyzed further in three-dimensional reconstructions using computer software. Additional apical Sertoli cell processes were probed for plectin and analyzed using conventional fluorescence microscopy. Antibodies generated against elements of the spectrin cytoskeleton react with material around and between the actin cuffs of tubulobulbar complexes, but appear excluded from the actin cuffs themselves. A similar staining pattern occurs with a probe for plectin. Immunoelectron microscopy confirmed the staining patterns observed by fluourescence microscopy. Based on our results, we suggest that a network of spectrin and plectin forms a scaffold around tubulobulbar complexes that may provide support for the actin network that cuffs each complex and also link adjacent complexes together.

Tubulobulbar complexes (TBCs) are actin‐related double membrane structures that are suspected to internalize intercellular junctions, a process that is crucial for junction turnover and sperm release. At apical sites of adhesion between spermatids and Sertoli cells, each TBC consists of a tubulobulbar projection of the spermatid plasma membrane, the surrounding attached plasma membrane of the adjacent Sertoli cell, and an actin network cuff. In other systems, spectrin is part of the membrane skeleton that links actin filaments to the plasma membrane. Others previously have reported that the protein is present at differentiating germ cells and epididymal spermatozoa. Plectin links cytoskeletal elements to each other and to the plasma membrane. We probed the seminiferous epithelium for spectrin, plectin and actin on fixed frozen sections of rat testis and fixed epithelial fragments. Both in sections and in epithelial fragments, beta‐spectrin II and plectin are localized to apical actin‐related tubulobulbar. Neither protein is concentrated at actin‐related intercellular adhesion junctions (ectoplasmic specializations). Our results demonstrate that both spectrin and plectin are present in Sertoli cells at tubulobulbar complexes. Supported by an NSERC Discovery Grant to AWV.Grant Funding Source: NSERC