Immunoelectron Microscopy of Endocytosis and Junction Proteins at Tubulobulbar Complexes in the Mammalian Testis

The seminiferous epithelium is the site of spermatogenesis, the cellular basis for male fertility. Large junction complexes separate the epithelium into basal and adluminal compartments, as well as attach morphologically differentiating spermatids to apical regions of Sertoli cells. During spermatogenesis, turnover of these large junction complexes enables both the next generation of spermatogenic cells to move from the basal into adluminal regions of the epithelium, and mature spermatozoa to be released into the duct system of the reproductive tract. We have proposed that unique structures termed tubulobulbar complexes (TBCs) are subcellular clathrin‐based endocytosis machines that internalize intact intercellular junctions during spermatid translocation and sperm release. If this is true, then proteins associated with endocytosis in general should be associated with TBCs. Previous immunofluorescence results have indicated that Rab5 and EEA1, together with junction proteins, are present in areas occupied by TBCs. However, association with specific compartments of TBCs at the ultrastructural level has not been done. Conventional methods of immunoelectron microscopy do not provide adequate preservation of membranes to clearly localize these proteins to TBCs. We have applied a recently published pre‐embedding protocol (Melo et al. 2014, Nature Protocols 9:2382‐2394) to the rat testis and show that Rab5 associates with the bulb region of TBCs. EEA1 is associated mainly with regions after the bulbs have been internalized. Nectin‐2 associates with junctions and with TBCs. This data supports the hypothesis that TBCs are involved with endocytosis of junction proteins. Support or Funding Information NSERC Discovery Grant to A. Wayne Vogl