Ca2+ signaling machinery is present at intercellular junctions and structures associated with junction turnover in rat Sertoli cells†

Abstract

The endoplasmic reticulum (ER) in Sertoli cells is a component of unique adhesion junctions (ectoplasmic specializations-ESs) and is closely associated with structures termed tubulobulbar complexes (TBCs) that internalize intercellular junctions during sperm release and during the translocation of spermatocytes through the blood-testis barrier. A role for the ER in Ca2+ regulation at ESs and TBCs has been suspected, but evidence for this function has proved elusive. Using electron microscopy, we define two new ER-plasma membrane (PM) contact sites in apical Sertoli cell processes. One of these sites occurs at TBCs where flattened lamellar cisternae of ER envelope the swollen bulb regions of the complexes, and where the gap between adjacent membranes is 12 nm. The other is at the periphery of apical processes where the gap between membranes is 13-14 nm. Using immunolocalization at the light and electron microscopic levels, we demonstrate that Ca2+ regulatory machinery is present at the ESs attached to spermatid heads, and at ER-PM contacts. Sarco/endoplasmic reticulum Ca2+-ATPase 2 (ATP2A2, SERCA2) is present at ESs; transient receptor potential channel subfamily M member 6 (TRPM6), Homer1 (HOMER1), and inositol 1,4,5-trisphosphate receptor (ITPR, IP3R) are present at ER-PM contacts associated with TBC bulbs; and stromal interacting molecule 1 (STIM1), Orai1 (ORAI1), and ATP2A2 are present at the ER-PM contacts around the margins of Sertoli cell apical processes. In Sertoli cells, the molecular machinery associated with ER generated Ca2+ fluxes is present in regions and structures directly related to junction remodeling-a process necessary for sperm release.

Multiple aspects of male germ cell development and interactions with Sertoli cells require inositol hexakisphosphate kinase-1

“…The apical ES is a dynamic structure, which normally organizes and orients the spermatids within the seminiferous epithelium. Fragmentation or malformation of the apical ES is associated with infertility 22 , 35 , 36 . The apical ES comprises multiple cytoskeleton proteins, adaptor proteins and signaling proteins 22 .…”

Alternative splicing switches STIM1 targeting to specialized membrane contact sites and modifies SOCE

“…S2). In serial sections of adult mouse testes, we reproduced the STIM1 distribution seen by Vogl et al in Sertoli cells 27 using a polyclonal antibody against the N-terminal domain of STIM1 ( Fig. 2b, for no-primary antibody controls see Fig.…”

“…2a), we noticed intense punctate immunoreactivity with the A antibody in apical regions of Sertoli cells, where spermatids are released and the ER forms specialized contacts to the plasma membrane within the so called tubulobulbar complexes (TBC). TBCs are unique to Sertoli cells and are clathrin-dependent subcellular structures required for internalization of intracellular junctions and membranes once spermatids are released 24,27,28 . At higher magnification, we observed that these punctate structures often contain a less stained lumen (arrows in Fig.…”

“…The intercellular adhesion with differentiating germ cells and between Sertoli cells are named "ectoplasmic specializations" (ES) and are defined as a link of ER cisternae to actin filaments, which in turn are linked to regions of the Sertoli cell plasma membrane. The mostly apically localized so-called "tubulobulbar complexes" (TBCs) are specialized ER regions internalizing intercellular junctions after sperm release 27 and have some molecular and structural similarities to podosomes of other cells 39 as well as sharing common features with clathrin-mediated endocytosis 40,41 . They also contain specialized membrane contact sites (MCS) devoid of actin which contain essential proteins of the lipid transfer machinery 42 (for schematic visualization see Fig.…”

Sticking With It: ER-PM Membrane Contact Sites as a Coordinating Nexus for Regulating Lipids and Proteins at the Cell Cortex

“…The distribution of cER along the PM can also vary in different polarized cell types. For instance, PM-associated cER is more abundant on basal membranes in HeLa cells though in Sertoli cells and polarized epithelia, ER-PM contacts occur at apical membranes (Orci et al, 2009;Son et al, 2016;Lyon et al, 2017). In neuron cell bodies the cER covers 12.5% of the PM inner surface, though only 0.5−1% of the PM contacts ER in axons (Wu et al, 2017;Chen et al, 2019).…”