TSPO x Tubulin

The outer mitochondrial membrane translocator protein (TSPO) binds cholesterol with high affinity and is involved in mediating its delivery into mitochondria, the rate-limiting step in hormone-induced steroidogenesis. Specific ligand binding to TSPO has been shown to initiate steroid formation. However, recent studies of the genetic deletion of Tspo have provided conflicting results. Here, we address and extend previous studies by examining the effects of Tspo-specific mutations on steroid formation in hormone- and cyclic adenosine monophosphate (cAMP)–responsive MA-10 cells, using the CRISPR/Cas9 system. Two mutant subcell lines, nG1 and G2G, each carrying a Tspo exon2-specific genome modification, and two control subcell lines, G1 and HH, each carrying a wild-type Tspo, were produced. In response to dibutyryl cAMP, the nG1 and G2G cells produced progesterone at levels significantly lower than those produced by the corresponding control cells G1 and HH. Neutral lipid homeostasis, which provides free cholesterol for steroid biosynthesis, was altered significantly in the Tspo mutant cells. Interestingly, the mitochondrial membrane potential (ΔΨm) of the Tspo mutant cells was significantly reduced compared with that of the control cells, likely because of TSPO interactions with the voltage-dependent anion channel and tubulin at the outer mitochondrial membrane. Steroidogenic acute regulatory protein (STAR) expression was induced in nG1 cells, suggesting that reduced TSPO affected STAR synthesis and/or processing. Taken together, these results provide further evidence for the critical role of TSPO in steroid biosynthesis and suggest that it may function at least in part via its regulation of ΔΨm and effects on STAR.

Immunocytochemistry is a compromise between maintaining antigenicity and preserving tissue morphology. In the testis, successful immunostaining results at the level of resolution provided by the light microscope have been obtained through use of either frozen or paraffin sections, although both techniques are fraught with limitations. With freezing, tissue preservation is not optimum, whereas with paraffin embedding, antigenicity is often destroyed. These limitations are not trivial and have led to numerous ambiguous results in the literature. In the present study we wish to report the results of immunocytochemical localization of various proteins in testis fixed by perfusion with Bouin's fluid and embedded in polyester wax, a ribboning embedding medium for histology. The advantages of this medium are that it does not require clearing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38 degrees C. Because of these two properties, the polyester was appears to adequately maintain antigenicity as compared to that observed in frozen sections, yet because it is a ribboning wax, it preserves detailed structure as well as paraffin does. Proteins that were immunolocalized included cytoskeletal proteins (tubulin, actin, vinculin, vimentin) and cell-specific markers: 1) androgen-binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiazepine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ cells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and both rabbit antisera and mouse monoclonal antibodies were used with equal success. In addition, fluorochrome-labeled second antibodies combined with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguously immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubulin or vimentin immunostaining patterns in Sertoli cells are altered during the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report that anti-PBR is a specific probe for Leydig cells in vivo and that an anti-nuclear lamin antibody appears to serve as a specific probe for spermatogonia and pachytene spermatocytes, but that the commercially available anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule.(ABSTRACT TRUNCATED AT 400 WORDS)