IP ₃ R is localized to ER‐PM contact sites at unique endocytic structures involved in intercellular junction turnover events critical to spermatogenesis.

Unique endocytic structures termed tubulobulbar complexes (TBCs) are critical for mammalian spermatogenesis. They function to internalize intercellular junctions located at the blood‐testis barrier basally in the seminiferous epithelium to allow spermatocytes to move from the basal compartment into the adluminal compartment. They also function to internalize apical attachments between spermatids and Sertoli cells prior to their release into the seminiferous tubule lumen. TBCs have three distinct regions: a proximal tubule, a bulb, and a short distal tubule. The tubular regions are cuffed by dendritic actin networks and the distal tubule is capped with a clathrin‐coated pit. The bulb region lacks an actin cuff, but is closely associated with cisternae of endoplasmic reticulum (ER). The function of this ER‐plasma membrane (PM) contact is unknown but we suspect it may be involved in calcium signaling. We previously used immunofluorescence to determine whether IP 3 R, a calcium channel in the ER that releases calcium, is present at these ER‐PM contacts. We previously probed cryo‐sections and epithelial fragments of perfusion‐fixed rat testes with antibodies against IP 3 R and an appropriate fluorescent secondary. Our previous data showed that IP 3 R labels the distal region of TBCs which is consistent with where we would expect the bulb region to be. To gather more convincing evidence, we used thicker cryo‐sections of 10 μm compared to our standard 5 μm to ensure that entire clusters of TBCs would be present in the section. We performed 3D reconstruction on deconvolved Z‐stacks to evaluate the localization of the IP 3 R labeling. The labeling is seen located between the distal and proximal tubules of TBCs indicating that it is at bulb region containing the ER‐PM contacts. To determine whether or not calcium release from IP 3 R channels occurs at these ER contacts, Adenophostin A, an IP 3 R agonist, was used to induce calcium release in isolated intact spermatids with associated Sertoli cell regions containing clusters of TBCs. Calcium release was monitored using Fluo4, a fluorescent calcium indicator. Our preliminary data suggests that Adenophostin A induces calcium release from IP 3 R channels in regions of isolated spermatids that is consistent with the bulb regions of TBCs. The data are consistent with our hypothesis that these ER‐PM contacts are involved in calcium signaling. Support or Funding Information Supported by a NSERC Discovery Grant (#155397‐2013) to AWV.

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